Manipulation of cellulose and/or β-1,4,-glucan

ABSTRACT

The present invention relates generally to isolated genes which encode polypeptides involved in cellulose biosynthesis in plants and transgenic plants expressing same in sense or antisense orientation, or as ribozymes, co-suppression or gene-targeting molecules. More particularly, the present invention is directed to a nucleic acid molecule isolated from  Arabidopsis thaliana, Oryza sativa , wheat, barley, maize,  Brassica  spp.,  Gossypium hirsutum  and  Eucalyptus  spp. which encode or an enzyme which is important in cellulose biosynthesis, in particular the cellulose synthase enzyme and homologues, analogues and derivatives thereof and uses of same in the production of transgenic plants expressing altered cellulose biosynthetic properties.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/221,013, filed Dec. 23, 1998, now U.S. Pat. No. 6,495,740 issued Dec.17, 2002, which is a continuation of International Patent ApplicationPCT/AU97/00402, filed Jun. 24, 1997, which application is incorporatedherein and which application claims priority to Australian PatentApplication PO 0699/96, filed Jun. 27, 1996.

The present invention relates generally to isolated genes which encodepolypeptides involved in cellulose biosynthesis and transgenic organismsexpressing same in sense or antisense orientation, or as ribozymes,co-suppression or gene-targeting molecules. More particularly, thepresent invention is directed to a nucleic acid molecule isolated fromArabidopsis thaliana, Oryza sativa, wheat, barley, maize, Brassica spp.,Gossypium hirsutum and Eucalyptus spp. which encode an enzyme which isimportant in cellulose biosynthesis, in particular the cellulosesynthase enzyme and homologues, analogues and derivatives thereof anduses of same in the production of transgenic plants expressing alteredcellulose biosynthetic properties.

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description. Sequenceidentity numbers (SEQ ID Nos.) for the nucleotide and amino acidsequences referred to in the specification are defined after thebibliography.

Throughout the specification, unless the context requires otherwise, theword “comprise”, or variations such as “comprises” or “comprising” willbe understood to imply the inclusion of a stated element or integer orgroup of elements or integers but not the exclusion of any other elementor integer or group of elements or integers.

Cellulose, the world's most abundant biopolymer, is the mostcharacteristic component of plant cell walls in so far as it forms muchof the structural framework of the cell wall. Cellulose is comprised ofcrystalline β-1,4-glucan microfibrils. The crystalline microfibrils areextremely strong and resist enzymic and mechanical degradation, animportant factor in determining the nutritional quantity, digestibilityand palatability of animal and human foodstuffs. As cellulose is alsothe dominant structural component of industrially-important plantfibres, such as cotton, flax, hemp, jute and the timber crops such asEucalyptus spp. and Pinus spp., amongst others, there is considerableeconomic benefit to be derived from the manipulation of cellulosecontent and/or quantity in plants. In particular, the production of foodand fibre crops with altered cellulose content is highly desirableobjectives.

The synthesis of cellulose involves the β-1,4-linkage of glucosemonomers, in the form of a nucleoside diphospoglucose such asUDP-glucose, to a pre-existing cellulose chain, catalysed by the enzymecellulose synthase.

Several attempts to identify the components of the functional cellulosesynthase in plants have failed, because levels of β-1,4-glucan orcrystalline cellulose produced in such assays have hitherto been too lowto permit enzyme purification for protein sequence determination.Insufficient homology between bacterial β-1,4-glucan synthase genes andplant cellulose synthase genes has also prevented the use ofhybridisation as an approach to isolating the plant homologues ofbacterial β-1,4-glucan (cellulose) synthases.

Furthermore, it has not been possible to demonstrate that the cellulosesynthase enzyme from plants is the same as, or functionally related to,other purified and characterised enzymes involved in polysaccharidebiosynthesis. As a consequence, the cellulose synthase enzyme has notbeen isolated from plants and, until the present invention, no nucleicacid molecule has been characterised which functionally-encodes a plantcellulose synthase enzyme.

In work leading up to the present invention, the inventors havegenerated several novel mutant Arabidopsis thaliana plants which aredefective in cellulose biosynthesis. The inventors have further isolateda cellulose synthase gene designated RSW1, which is involved incellulose biosynthesis in Arabidopsis thaliana, and homologous sequencesin Oryza sativa, wheat, barley, maize, Brassica spp., Gossypium hirsutumand Eucalyptus spp. The isolated nucleic acid molecules of the presentinvention provide the means by which cellulose content and structure maybe modified in plants to produce a range of useful fibres suitable forspecific industrial purposes, for example increased decay resistance oftimber and altered digestibility of foodstuffs, amongst others.

Accordingly, one aspect of the present invention provides an isolatednucleic acid molecule comprising a sequence of nucleotides whichencodes, or is complementary to a sequence which encodes a polypeptideof the cellulose biosynthetic pathway or a functional homologue,analogue or derivative thereof.

The nucleic acid molecule of the invention may be derived from aprokaryotic source or an eukaryotic source.

Those skilled in the art will be aware that cellulose productionrequires not only the presence of a catalytic subunit, but also itsactivation and organisation into arrays which favour the crystallizationof glucan chains. This organisation is radically different betweenbacteria, which possess linear arrays, and higher plants, which possesshexameric clusters or “rosettes”, of glucan chains. The correctorganisation and activation of the bacterial enzyme may require manyfactors which are either not known, or alternatively, not known to bepresent in plant cells, for example specific membrane lipids to impartan active conformation on the enzyme complex or protein, or thebacterial c-di-GMP activation system. Accordingly, the use of aplant-derived sequence in eukaryotic cells such as plants providessignificant advantages compared to the use of bacterially-derivedsequences.

Accordingly, the present invention does not extend to known genesencoding the catalytic subunit of Agrobacterium tumefaciens orAcetobacter xylinum or Acetobacter pasteurianus cellulose synthase, orthe use of such known bacterial genes and polypeptides to manipulatecellulose.

Preferably, the subject nucleic acid molecule is derived from aneukaryotic organism.

In a more preferred embodiment of the invention, the isolated nucleicacid molecule of the invention encodes a plant cellulose synthase or acatalytic subunit thereof, or a homologue, analogue or derivativethereof.

More preferably, the isolated nucleic acid molecule encodes a plantcellulose synthase polypeptide which is associated with the primary cellwall of a plant cell. In an alternative preferred embodiment, thenucleic acid molecule of the invention encodes a plant cellulosesynthase or catalytic subunit thereof which is normally associated withthe secondary cell wall of a plant cell.

In a more preferred embodiment, the nucleic acid molecule of theinvention is a cDNA molecule, genomic clone, mRNA molecule or asynthetic oligonucleotide molecule.

In a particularly preferred embodiment, the present invention providesan isolated nucleic acid molecule which encodes or is complementary to anucleic acid molecule which encodes the Arabidopsis thaliana, Gossypiumhirsutum (cotton), Oryza sativa (rice), Eucalyptus spp., Brassica spp.wheat, barley or maize cellulose synthase enzyme or a catalytic subunitthereof or a polypeptide component, homologue, analogue or derivativethereof.

As exemplified herein, the present inventors have identified cellulosebiosynthesis genes in maize, wheat, barley, rice, cotton, Brassica spp.and Eucalyptus spp., in addition to the specific Arabidopsis thalianaRSW1 gene sequence which has been shown to be particularly useful foraltering cellulose and/or β-1,4-glucan and/or starch levels in cells.

Hereinafter the term “polypeptide of the cellulose biosynthetic pathway”or similar term shall be taken to refer to a polypeptide or a protein ora part, homologue, analogue or derivative thereof which is involved inone or more of the biosynthetic steps leading to the production ofcellulose or any related β-1,4-glucan polymer in plants. In the presentcontext, a polypeptide of the cellulose biosynthetic pathway shall alsobe taken to include both an active enzyme which contributes to thebiosynthesis of cellulose or any related β-1,4-glucan polymer in plantsand to a polypeptide component of such an enzyme. As used herein, apolypeptide of the cellulose biosynthetic pathway thus includescellulose synthase. Those skilled in the art will be aware of othercellulose biosynthetic pathway polypeptides in plants.

The term “related β-1,4-glucan polymer” shall be taken to include anycarbohydrate molecule comprised of a primary structure of β-1,4-linkedglucose monomers similar to the structure of the components of thecellulose microfibril, wherein the relative arrangement or relativeconfiguration of the glucan chains may differ from their relativeconfiguration in microfibrils of cellulose. As used herein, a relatedβ-1,4-glucan polymer includes those β-1,4-glucan polymers whereinindividual β-1,4-glucan microfibrils are arranged in an anti-parallel orsome other relative configuration not found in a cellulose molecule ofplants and those non-crystalline β-1,4-glucans described as lacking theresistance to extraction and degradation that characterise cellulosemicrofibrils.

The term “cellulose synthase” shall be taken to refer to a polypeptidewhich is required to catalyse a β-1,4-glucan linkage to a cellulosemicrofibril.

Reference herein to “gene” is to be taken in its broadest context andincludes:

-   -   (i) a classical genomic gene consisting of transcriptional        and/or translational regulatory sequences and/or a coding region        and/or non-translated sequences (i.e. introns, 5′- and        3′-untranslated sequences); or    -   (ii) mRNA or cDNA corresponding to the coding regions (i.e.        exons) and 5′- and 3′-untranslated sequences of the gene.

The term “gene” is also used to describe synthetic or fusion moleculesencoding all or part of a functional product.

In the present context, the term “cellulose gene” or “cellulose geneticsequence” or similar term shall be taken to refer to any gene ashereinbefore defined which encodes a polypeptide of the cellulosebiosynthetic pathway and includes a cellulose synthase gene.

The term “cellulose synthase gene” shall be taken to refer to anycellulose gene which specifically encodes a polypeptide which is acomponent of a functional enzyme having cellulose synthase activity i.e.an enzyme which catalyses a β-1,4-glucan linkage to a cellulosemicrofibril.

Preferred cellulose genes may be derived from a naturally-occurringcellulose gene by standard recombinant techniques. Generally, acellulose gene may be subjected to mutagenesis to produce single ormultiple nucleotide substitutions, deletions and/or additions.Nucleotide insertional derivatives of the cellulose synthase gene of thepresent invention include 5′ and 3′ terminal fusions as well asintra-sequence insertions of single or multiple nucleotides. Insertionalnucleotide sequence variants are those in which one or more nucleotidesare introduced into a predetermined site in the nucleotide sequencealthough random insertion is also possible with suitable screening ofthe resulting product. Deletional variants are characterised by theremoval of one or more nucleotides from the sequence. Substitutionalnucleotide variants are those in which at least one nucleotide in thesequence has been removed and a different nucleotide inserted in itsplace. Such a substitution may be “silent” in that the substitution doesnot change the amino acid defined by the codon. Alternatively,substituents are designed to alter one amino acid for another similaracting amino acid, or amino acid of like charge, polarity, orhydrophobicity.

As used herein, the term “derived from” shall be taken to indicate thata particular integer or group of integers has originated from thespecies specified, but has not necessarily been obtained directly fromthe specified source.

For the present purpose, “homologues” of a nucleotide sequence shall betaken to refer to an isolated nucleic acid molecule which issubstantially the same as the nucleic acid molecule of the presentinvention or its complementary nucleotide sequence, notwithstanding theoccurrence within said sequence, of one or more nucleotidesubstitutions, insertions, deletions, or rearrangements.

“Analogues” of a nucleotide sequence set forth herein shall be taken torefer to an isolated nucleic acid molecule which is substantially thesame as a nucleic acid molecule of the present invention or itscomplementary nucleotide sequence, notwithstanding the occurrence of anynon-nucleotide constituents not normally present in said isolatednucleic acid molecule, for example carbohydrates, radiochemicalsincluding radionucleotides, reporter molecules such as, but not limitedto DIG, alkaline phosphatase or horseradish peroxidase, amongst others.

“Derivatives” of a nucleotide sequence set forth herein shall be takento refer to any isolated nucleic acid molecule which containssignificant sequence similarity to said sequence or a part thereof.Generally, the nucleotide sequence of the present invention may besubjected to mutagenesis to produce single or multiple nucleotidesubstitutions, deletions and/or insertions. Nucleotide insertionalderivatives of the nucleotide sequence of the present invention include5′ and 3′ terminal fusions as well as intra-sequence insertions ofsingle or multiple nucleotides or nucleotide analogues. Insertionalnucleotide sequence variants are those in which one or more nucleotidesor nucleotide analogues are introduced into a predetermined site in thenucleotide sequence of said sequence, although random insertion is alsopossible with suitable screening of the resulting product beingperformed. Deletional variants are characterised by the removal of oneor more nucleotides from the nucleotide sequence. Substitutionalnucleotide variants are those in which at least one nucleotide in thesequence has been removed and a different nucleotide or nucleotideanalogue inserted in its place.

The present invention extends to the isolated nucleic acid molecule whenintegrated into the genome of a cell as an addition to the endogenouscellular complement of cellulose synthase genes. The said integratednucleic acid molecule may, or may not, contain promoter sequences toregulate expression of the subject genetic sequence.

The isolated nucleic acid molecule of the present invention may beintroduced into and expressed in any cell, for example a plant cell,fungal cell, insect cell. animal cell, yeast cell or bacterial cell.Those skilled in the art will be aware of any modifications which arerequired to the codon usage or promoter sequences or other regulatorysequences, in order for expression to occur in such cells.

Another aspect of the present invention is directed to a nucleic acidmolecule which comprises a sequence of nucleotides corresponding orcomplementary to any one or more of the sequences set forth in SEQ IDNos:1, 3, 4, 5, 7, 9, 11, or 13, or having at least about 40%, morepreferably at least about 55%, still more preferably at least about 65%,yet still more preferably at least about 75–80% and even still morepreferably at least about 85–95% nucleotide similarity to all, or a partthereof.

According to this aspect of the invention, said nucleic acid moleculeencodes, or is complementary to a nucleotide sequence encoding, apolypeptide of the cellulose biosynthetic pathway in a plant or ahomologue, analogue or derivative thereof.

Preferably, a nucleic acid molecule which is at least 40% related to anyone or more of the sequences set forth in SEQ ID Nos:1, 3, 4, 5, 7, 9,11, or 13 comprises a nucleotide sequence which encodes or iscomplementary to a sequence which encodes a plant cellulose synthase,more preferably a cellulose synthase which is associated with theprimary or the secondary plant cell wall of the species from which ithas been derived.

Furthermore, the nucleic acid molecule according to this aspect of theinvention may be derived from a monocotyledonous or dicotyledonous plantspecies. In a particularly preferred embodiment, the nucleic acidmolecule is derived from Arabidopsis thaliana, Oryza sativa, wheat,barley, maize, Brassica spp., Gossypium hirsutum (cotton) or Eucalyptusspp., amongst others.

For the purposes of nomenclature, the nucleotide sequence shown in SEQID NO:1 relates to a cellulose gene as hereinbefore defined whichcomprises a cDNA sequence designated T20782 and which is derived fromArabidopsis thaliana. The amino acid sequence set forth in SEQ ID NO:2relates to the polypeptide encoded by T20782.

The nucleotide sequence set forth in SEQ ID NO:3 relates to thenucleotide sequence of the complete Arabidopsis thaliana genomic geneRSW1, including both intron and exon sequences. The nucleotide sequenceof SEQ ID NO:3 comprises exons 1–14 of the genomic gene and includes2295 bp of 5′-untranslated sequences, of which approximately the first1.9 kb comprises RSW1 promoter sequence (there is a putative TATA boxmotif at positions 1843–1850 of SEQ ID NO:3). The nucleotide sequenceset forth in SEQ ID NO:3 is derived from the cosmid clone 23H12. Thissequence is also the genomic gene equivalent of SEQ ID Nos:1 and 5.

The nucleotide sequence set forth in SEQ ID NO:4 relates to the partialnucleotide sequence of a genomic gene variant of RSW1, derived fromcosmid clone 12C4. The nucleotide sequence of SEQ ID NO:4 comprises exonsequence 1–11 and part of exon 12 of the genomic gene sequence andincludes 862 bp of 5′-untranslated sequences, of which approximately 700nucleotides comprise RSW1 promoter sequences (there is a putative TATAbox motif at positions 668–673 of SEQ ID NO:4). The genomic genesequence set forth in SEQ ID NO:4 is the equivalent of the cDNA sequenceset forth in SEQ ID NO:7 (i.e. eDNA clone Ath-A).

The nucleotide sequence shown in SEQ ID NO:5 relates to a cellulose geneas hereinbefore defined which comprises a cDNA equivalent of theArabidopsis thaliana RSW1 gene set forth in SEQ ID NO:3. The amino acidsequence set forth in SEQ ID NO:6 relates to the polypeptide encoded bythe wild-type RSW1 gene sequences set forth in SEQ ID Nos:3 and 5.

The nucleotide sequence shown in SEQ ID NO:7 relates to a cellulose geneas hereinbefore defined which comprises a cDNA equivalent of theArabidopsis thaliana RSW1 gene set forth in SEQ ID NO:4. The nucleotidesequence is a variant of the nucleotide sequences set forth in SEQ IDNos:3 and 5. The amino acid sequence set forth in SEQ ID NO:8 relates tothe polypeptide encoded by the wild-type RSW1 gene sequences set forthin SEQ ID Nos:4 and 6.

The nucleotide sequence shown in SEQ ID NO:9 relates to a cellulose geneas hereinbefore defined which comprises a further wild-type variant ofthe Arabidopsis thaliana RSW1 gene set forth in SEQ ID Nos:3 and 5. Thenucleotide sequence variant is designated Ath-B. The amino acid sequenceset forth in SEQ ID NO:10 relates to the polypeptide encoded by thewild-type RSW1 gene sequence set forth in SEQ ID No:9.

The nucleotide sequence shown in SEQ ID NO:11 relates to a cellulosegene as hereinbefore defined which comprises a cDNA equivalent of theArabidopsis thaliana rsw1 gene. The rsw1 gene is a mutant cellulose genewhich produces a radial root swelling phenotype as described by Baskinet al.(1992). The present inventors have shown herein that the rsw1 genealso produces reduced inflorescence length, reduced fertility, misshapenepidermal cells, reduced cellulose content and the accumulation ofnon-crystalline β-1,4-glucan, amongst others, when expressed in plantcells. The rsw1 nucleotide sequence is a further variant of thenucleotide sequences set forth in SEQ ID Nos:3 and 5. The amino acidsequence set forth in SEQ ID NO:12 relates to the rsw1 polypeptideencoded by the mutant rsw1 gene sequence set forth in SEQ ID No:11.

The nucleotide sequence shown in SEQ ID NO:13 relates to a cellulosegene as hereinbefore defined which comprises a cDNA equivalent of theOryza sativa RSW1 or RSW1-like gene. The nucleotide sequence isclosely-related to the Arabidopsis thaliana RSW1 and rsw1 nucleotidesequences set forth herein (SEQ ID Nos:1, 3, 4, 5, 7, 9 and 11). Theamino acid sequence set forth in SEQ ID NO:14 relates to the polypeptideencoded by the RSW1 or RSW1-like gene sequences set forth in SEQ IDNo:13.

Those skilled in the art will be aware of procedures for the isolationof further cellulose genes to those specifically described herein, forexample further cDNA sequences and genomic gene equivalents, whenprovided with one or more of the nucleotide sequences set forth in SEQID Nos:1, 3, 4, 5, 7, 9, 11, or 13. In particular, hybridisations may beperformed using one or more nucleic acid hybridisation probes comprisingat least 10 contiguous nucleotides and preferably at least 50 contiguousnucleotides derived from the nucleotide sequences set forth herein, toisolate cDNA clones, mRNA molecules, genomic clones from a genomiclibrary (in particular genomic clones containing the entire 5′ upstreamregion of the gene including the promoter sequence, and the entirecoding region and 3′-untranslated sequences), and/or syntheticoligonucleotide molecules, amongst others. The present invention clearlyextends to such related sequences.

The invention further extends to any homologues, analogues orderivatives of any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13.

A further aspect of the present invention contemplates a nucleic acidmolecule which encodes or is complementary to a nucleic acid moleculewhich encodes, a polypeptide which is required for cellulosebiosynthesis in a plant, such as cellulose synthase, and which iscapable of hybridising under at least low stringency conditions to thenucleic acid molecule set forth in any one or more of SEQ ID Nos:1, 3,4, 5, 7, 9, 11 or 13, or to a complementary strand thereof.

As an exemplification of this embodiment, the present inventors haveshown that it is possible to isolate variants of the Arabidopsisthaliana RSW1 gene sequence set forth in SEQ ID NO:3, by hybridizationunder low stringency conditions. Such variants include related sequencesderived from Gossypium hirsutum (cotton), Eucalyptus spp. and A.thaliana. Additional variant are clearly encompassed by the presentinvention.

Preferably, the nucleic acid molecule further comprises a nucleotidesequence which encodes, or is complementary to a nucleotide sequencewhich encodes, a cellulose synthase polypeptide, more preferably acellulose synthase which is associated with the primary or secondaryplant cell wall of the plant species from which said nucleic acidmolecule was derived.

More preferably, the nucleic acid molecule according to this aspect ofthe invention encodes or is complementary to a nucleic acid moleculewhich encodes, a polypeptide which is required for cellulosebiosynthesis in a plant, such as cellulose synthase, and which iscapable of hybridising under at least medium stringency conditions tothe nucleic acid molecule set forth in any one or more of SEQ ID Nos:1,3, 4, 5, 7, 9, 11 or 13, or to a complementary strand thereof.

Even more preferably, the nucleic acid molecule according to this aspectof the invention encodes or is complementary to a nucleic acid moleculewhich encodes, a polypeptide which is required for cellulosebiosynthesis in a plant, such as cellulose synthase, and which iscapable of hybridising under at least high stringency conditions to thenucleic acid molecule set forth in any one or more of SEQ ID Nos:1, 3,4, 5, 7, 9, 11 or 13, or to a complementary strand thereof.

For the purposes of defining the level of stringency, a low stringencyis defined herein as being a hybridisation and/or a wash carried out in6×SSC buffer, 0.1% (w/v) SDS at 28° C. Generally, the stringency isincreased by reducing the concentration of SSC buffer, and/or increasingthe concentration of SDS and/or increasing the temperature of thehybridisation and/or wash. A medium stringency comprises a hybridisationand/or a wash carried out in 0.2×SSC–2×SSC buffer, 0.1% (w/v) SDS at 42°C. to 65° C., while a high stringency comprises a hybridisation and/or awash carried out in 0.1×SSC–0.2×SSC buffer, 0.1% (w/v) SDS at atemperature of at least 55° C. Conditions for hybridisations and washesare well understood by one normally skilled in the art. For the purposesof further clarification only, reference to the parameters affectinghybridisation between nucleic acid molecules is found in pages 2.10.8 to2.10.16. of Ausubel et al. (1987), which is herein incorporated byreference.

In an even more preferred embodiment of the invention, the isolatednucleic acid molecule further comprises a sequence of nucleotides whichis at least 40% identical to at least 10 contiguous nucleotides derivedfrom any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or acomplementary strand thereof.

Still more preferably, the isolated nucleic acid molecule furthercomprises a sequence of nucleotides which is at least 40% identical toat least 50 contiguous nucleotides derived from the sequence set forthin any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or acomplementary strand thereof.

The present invention is particularly directed to a nucleic acidmolecule which is capable of functioning as a cellulose gene ashereinbefore defined, for example a cellulose synthase gene such as, butnot limited to, the Arabidopsis thaliana, Oryza sativa, wheat, barley,maize, Brassica spp., Gossypium hirsutum or Eucalyptus spp. cellulosesynthase genes, amongst others. The subject invention clearlycontemplates additional cellulose genes to those specifically describedherein which are derived from these plant species.

The invention further contemplates other sources of cellulose genes suchas but not limited to, tissues and cultured cells of plant origin.Preferred plant species according to this embodiment include hemp, jute,flax and woody plants including, but not limited to Pinus spp., Populusspp., Picea spp., amongst others.

A genetic sequence which encodes or is complementary to a sequence whichencodes a polypeptide which is involved in cellulose biosynthesis maycorrespond to the naturally occurring sequence or may differ by one ormore nucleotide substitutions, deletions and/or additions. Accordingly,the present invention extends to cellulose genes and any functionalgenes, mutants, derivatives, parts, fragments, homologues or analoguesthereof or non-functional molecules but which are at least useful as,for example, genetic probes, or primer sequences in the enzymatic orchemical synthesis of said gene, or in the generation of immunologicallyinteractive recombinant molecules.

In a particularly preferred embodiment, the cellulose genetic sequencesare employed to identify and isolate similar genes from plant cells,tissues, or organ types of the same species, or from the cells, tissues,or organs of another plant species.

According to this embodiment, there is contemplated a method foridentifying a related cellulose gene or related cellulose geneticsequence, for example a cellulose synthase or cellulose synthase-likegene, said method comprising contacting genomic DNA, or mRNA, or cDNAwith a hybridisation effective amount of a first cellulose geneticsequence comprising any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11or 13, or a complementary sequence, homologue, analogue or derivativethereof derived from at least 10 contiguous nucleotides of said firstsequence, and then detecting said hybridisation.

Preferably, the first genetic sequence comprises at least 50 contiguousnucleotides, even more preferably at least 100 contiguous nucleotidesand even more preferably at least 500 contiguous nucleotides, derivedfrom any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or acomplementary strand, homologue, analogue or derivative thereof.

The related cellulose gene or related cellulose genetic sequence may bein a recombinant form, in a virus particle, bacteriophage particle,yeast cell, animal cell, or a plant cell. Preferably, the relatedcellulose gene or related cellulose genetic sequence is derived from aplant species, such as a monocotyledonous plant or a dicotyledonousplant selected from the list comprising Arabidopsis thaliana, wheat,barley, maize, Brassica spp., Gossypium hirsutum (cotton), Oryza sativa(rice), Eucalyptus spp., hemp, jute, flax, and woody plants including,but not limited to Pinus spp., Populus spp., Picea spp., amongst others.

More preferably, related cellulose gene or related cellulose geneticsequence is derived from a plant which is useful in the fibre or timberindustries, for example Gossypium hirsutum (cotton), hemp, jute, flax,Eucalyptus spp. or Pinus spp., amongst others. Alternatively, therelated cellulose gene or related cellulose genetic sequence is derivedfrom a plant which is useful in the cereal or starch industry, forexample wheat, barley, rice or maize, amongst others.

In a particularly preferred embodiment, the first cellulose geneticsequence is labeled with a reporter molecule capable of giving anidentifiable signal (e.g. a radioisotope such as ³²P or ³⁵S or abiotinylated molecule).

An alternative method contemplated in the present invention involveshybridising two nucleic acid “primer molecules” to a nucleic acid“template molecule” which comprises a related cellulose gene or relatedcellulose genetic sequence or a functional part thereof, wherein thefirst of said primers comprises contiguous nucleotides derived from anyone or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13 or a homologue,analogue or derivative thereof and the second of said primers comprisescontiguous nucleotides complementary to any one or more of SEQ ID Nos:1,3, 4, 5, 7, 9, 11 or 13. Specific nucleic acid molecule copies of thetemplate molecule are amplified enzymatically in a polymerase chainreaction, a technique that is well known to one skilled in the art.

In a preferred embodiment, each nucleic acid primer molecule is at least10 nucleotides in length, more preferably at least 20 nucleotides inlength, even more preferably at least 30 nucleotides in length, stillmore preferably at least 40 nucleotides in length and even still morepreferably at least 50 nucleotides in length.

Furthermore, the nucleic acid primer molecules consists of a combinationof any of the nucleotides adenine, cytidine, guanine, thymidine, orinosine, or functional analogues or derivatives thereof which are atleast capable of being incorporated into a polynucleotide moleculewithout having an inhibitory effect on the hybridisation of said primerto the template molecule in the environment in which it is used.

Furthermore, one or both of the nucleic acid primer molecules may becontained in an aqueous mixture of other nucleic acid primer molecules,for example a mixture of degenerate primer sequences which vary fromeach other by one or more nucleotide substitutions or deletions.Alternatively, one or both of the nucleic acid primer molecules may bein a substantially pure form.

The nucleic acid template molecule may be in a recombinant form, in avirus particle, bacteriophage particle, yeast cell, animal cell, or aplant cell. Preferably, the nucleic acid template molecule is derivedfrom a plant cell, tissue or organ, in particular a cell, tissue ororgan derived from a plant selected from the list comprising Arabidopsisthaliana, Oryza sativa, wheat, barley, maize, Brassica spp., Gossypiumhirsutum and Eucalyptus spp., hemp, jute, flax, and woody plantsincluding, but not limited to Pinus spp., Populus spp., Picea spp.,amongst others.

Those skilled in the art will be aware that there are many knownvariations of the basic polymerase chain reaction procedure, which maybe employed to isolate a related cellulose gene or related cellulosegenetic sequence when provided with the nucleotide sequences set forthin any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13. Suchvariations are discussed, for example, in McPherson et al.(1991). Thepresent invention extends to the use of all such variations in theisolation of related cellulose genes or related cellulose geneticsequences using the nucleotide sequences embodied by the presentinvention.

The isolated nucleic acid molecule according to any of the furtherembodiments may be cloned into a plasmid or bacteriophage molecule, forexample to facilitate the preparation of primer molecules orhybridisation probes or for the production of recombinant gene products.Methods for the production of such recombinant plasmids, cosmids,bacteriophage molecules or other recombinant molecules are well-known tothose of ordinary skill in the art and can be accomplished without undueexperimentation. Accordingly, the invention further extends to anyrecombinant plasmid, bacteriophage, cosmid or other recombinant moleculecomprising the nucleotide sequence set forth in any one or more of SEQID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a complementary sequence,homologue, analogue or derivative thereof.

The nucleic acid molecule of the present invention is also useful fordeveloping genetic constructs which express a cellulose geneticsequence, thereby providing for the increased expression of genesinvolved in cellulose biosynthesis in plants, selected for example fromthe list comprising Arabidopsis thaliana, Oryza sativa, wheat, barley,maize, Brassica spp., Gossypium hirsutum and Eucalyptus spp., hemp,jute, flax, and woody plants including, but not limited to Pinus spp.,Populus spp., Picea spp., amongst others. The present inventionparticularly contemplates the modification of cellulose biosynthesis incotton, hemp, jute, flax, Eucalyptus spp. and Pinus spp., amongstothers.

The present inventors have discovered that the genetic sequencesdisclosed herein are capable of being used to modify the level ofnon-crystalline β-1,4,-glucan, in addition to altering cellulose levelswhen expressed, particularly when expressed in plants cells. Inparticular, the Arabidopsis thaliana rsw1 mutant has increased levels ofnon-crystalline β-1,4,-glucan, when grown at 31° C., compared towild-type plants, grown under identical conditions. The expression of agenetic sequence described herein in the antisense orientation intransgenic plants grown at only 21° C. is shown to reproduce manyaspects of the rsw1 mutant phenotype.

Accordingly, the present invention clearly extends to the modificationof non-crystalline β-1,4,-glucan biosynthesis in plants, selected forexample from the list comprising Arabidopsis thaliana, Oryza sativa,wheat, barley, maize, Brassica spp., Gossypium hirsutum and Eucalyptusspp., hemp, jute, flax, and woody plants including, but not limited toPinus spp., Populus spp., Picea spp., amongst others. The presentinvention particularly contemplates the modification of non-crystallineβ-1,4,-glucan biosynthesis in cotton, hemp, jute, flax, Eucalyptus spp.and Pinus spp., amongst others.

The present invention further extends to the production and use ofnon-crystalline β-1,4-glucan and to the use of the glucan to modify theproperties of plant cell walls or cotton fibres or wood fibres. Suchmodified properties are described herein (Example 13).

The inventors have discovered that the rsw1 mutant has altered carbonpartitioning compared to wild-type plants, resulting in significantlyhigher starch levels therein. The isolated nucleic acid moleculesprovided herein are further useful for altering the carbon partitioningin a cell. In particular, the present invention contemplates increasedstarch production in transgenic plants expressing the nucleic acidmolecule of the invention in the antisense orientation or alternatively,expressing a ribozyme or co-suppression molecule comprising the nucleicacid sequence of the invention.

The invention further contemplates reduced starch and/or non-crystallineβ-1,4-glucan product in transgenic plants expressing the nucleic acidmolecule of the invention in the sense orientation such that celluloseproduction is increased therein.

Wherein it is desired to increase cellulose production in a plant cell,the coding region of a cellulose gene is placed operably behind apromoter, in the sense orientation, such that a cellulose gene productis capable of being expressed under the control of said promotersequence. In a preferred embodiment, the cellulose genetic sequence is acellulose synthase genomic sequence, cDNA molecule or protein-codingsequence.

In a particularly preferred embodiment, the cellulose genetic sequencecomprises a sequence of nucleotides substantially the same as thesequence set forth in any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11or 13 or a homologue, analogue or derivative thereof.

Wherein it is desirable to reduce the content of cellulose or toincrease the content of non-crystalline β-1,4-glucan, the nucleic acidmolecule of the present invention is expressed in the antisenseorientation under the control of a suitable promoter. Additionally, thenucleic acid molecule of the invention is also useful for developingribozyme molecules, or in co-suppression of a cellulose gene. Theexpression of an antisense, ribozyme or co-suppression moleculecomprising a cellulose gene, in a cell such as a plant cell, fungalcell, insect cell. animal cell, yeast cell or bacterial cell, may alsoincrease the solubility, digestibility or extractability of metabolitesfrom plant tissues or alternatively, or increase the availability ofcarbon as a precursor for any secondary metabolite other than cellulose(e.g. starch or sucrose). By targeting the endogenous cellulose gene,expression is diminished, reduced or otherwise lowered to a level thatresults in reduced deposition of cellulose in the primary or secondarycell walls of the plant cell, fungal cell, insect cell. animal cell,yeast cell or bacterial cell, and more particularly, a plant cell.Additionally, or alternatively, the content of non-crystallineβ-1,4-glucan is increased in such cells.

Co-suppression is the reduction in expression of an endogenous gene thatoccurs when one or more copies of said gene, or one or more copies of asubstantially similar gene are introduced into the cell. The presentinvention also extends to the use of co-suppression to inhibit theexpression of a gene which encodes a cellulose gene product, such as butnot limited to cellulose synthase. Preferably, the co-suppressionmolecule of the present invention targets a plant mRNA molecule whichencodes a cellulose synthase enzyme, for example a plant, fungus, orbacterial cellulose synthase mRNA, and more preferably a plant mRNAderived from Arabidopsis thaliana, Oryza sativa, wheat, barley, maize,Brassica spp., Gossypium hirsutum and Eucalyptus spp., hemp, jute, flax,or a woody plant such as Pinus spp., Populus spp., or Picea spp.,amongst others.

In a particularly preferred embodiment, the gene which is targeted by aco-suppression molecule, comprises a sequence of nucleotides set forthin any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or acomplement, homologue, analogue or derivative thereof.

In the context of the present invention, an antisense molecule is an RNAmolecule which is transcribed from the complementary strand of a nucleargene to that which is normally transcribed to produce a “sense” mRNAmolecule capable of being translated into a polypeptide component of thecellulose biosynthetic pathway. The antisense molecule is thereforecomplementary to the mRNA transcribed from a sense cellulose gene or apart thereof. Although not limiting the mode of action of the antisensemolecules of the present invention to any specific mechanism, theantisense RNA molecule possesses the capacity to form a double-strandedmRNA by base pairing with the sense mRNA, which may prevent translationof the sense mRNA and subsequent synthesis of a polypeptide geneproduct.

Preferably, the antisense molecule of the present invention targets aplant mRNA molecule which encodes a cellulose gene product, for examplecellulose synthase. Preferably, the antisense molecule of the presentinvention targets a plant mRNA molecule which encodes a cellulosesynthase enzyme, for example a plant mRNA derived from Arabidopsisthaliana, Oryza sativa, wheat, barley, maize, Brassica spp., Gossypiumhirsutum and Eucalyptus spp., hemp, jute, flax, or a woody plant such asPinus spp., Populus spp., or Picea spp., amongst others.

In a particularly preferred embodiment, the antisense molecule of theinvention targets an mRNA molecule encoded by any one or more of SEQ IDNos:1, 3, 4, 5, 7, 9, 11 or 13, or a homologue, analogue or derivativethereof.

Ribozymes are synthetic RNA molecules which comprise a hybridisingregion complementary to two regions, each of at least 5 contiguousnucleotide bases in the target sense mRNA. In addition, ribozymespossess highly specific endoribonuclease activity, whichautocatalytically cleaves the target sense mRNA. A complete descriptionof the function of ribozymes is presented by Haseloff and Gerlach (1988)and contained in International Patent Application No. WO89/05852.

The present invention extends to ribozyme which target a sense mRNAencoding a cellulose gene product, thereby hybridising to said sensemRNA and cleaving it, such that it is no longer capable of beingtranslated to synthesise a functional polypeptide product. Preferably,the ribozyme molecule of the present invention targets a plant mRNAmolecule which encodes a cellulose synthase enzyme, for example a plantmRNA derived from Arabidopsis thaliana, Gossypium hirsutum (cotton),Oryza sativa (rice), Eucalyptus spp., hemp, jute, flax, or a woody plantsuch as Pinus spp., Populus spp., or Picea spp., amongst others.

In a particularly preferred embodiment, the ribozyme molecule willtarget an mRNA encoded by any one or more of SEQ ID Nos:1, 3, 4, 5, 7,9, 11 or 13, or a homologue, analogue or derivative thereof.

According to this embodiment, the present invention provides a ribozymeor antisense molecule comprising at least 5 contiguous nucleotide basesderived from any one or more of SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13,or a complementary nucleotide sequence or a homologue, analogue orderivative thereof, wherein said antisense or ribozyme molecule is ableto form a hydrogen-bonded complex with a sense mRNA encoding a cellulosegene product to reduce translation thereof.

In a preferred embodiment, the antisense or ribozyme molecule comprisesat least 10 to 20 contiguous nucleotides derived from any one or more ofSEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a complementary nucleotidesequence or a homologue, analogue or derivative thereof. Although thepreferred antisense and/or ribozyme molecules hybridise to at leastabout 10 to 20 nucleotides of the target molecule, the present inventionextends to molecules capable of hybridising to at least about 50–100nucleotide bases in length, or a molecule capable of hybridising to afull-length or substantially full-length mRNA encoded by a cellulosegene, such as a cellulose synthase gene.

Those skilled in the art will be aware of the necessary conditions, ifany, for selecting or preparing the antisense or ribozyme molecules ofthe invention.

It is understood in the art that certain modifications, includingnucleotide substitutions amongst others, may be made to the antisenseand/or ribozyme molecules of the present invention, without destroyingthe efficacy of said molecules in inhibiting the expression of a geneencoding a cellulose gene product such as cellulose synthase. It istherefore within the scope of the present invention to include anynucleotide sequence variants, homologues, analogues, or fragments of thesaid gene encoding same, the only requirement being that said nucleotidesequence variant, when transcribed, produces an antisense and/orribozyme molecule which is capable of hybridising to a sense mRNAmolecule which encodes a cellulose gene product.

Gene targeting is the replacement of an endogenous gene sequence withina cell by a related DNA sequence to which it hybridises, therebyaltering the form and/or function of the endogenous gene and thesubsequent phenotype of the cell. According to this embodiment, at leasta part of the DNA sequence defined by any one or more of SEQ ID Nos:1,3, 4, 5, 7, 9, 11 or 13, or a related cellulose genetic sequence, may beintroduced into target cells containing an endogenous cellulose gene,thereby replacing said endogenous cellulose gene.

According to this embodiment, the polypeptide product of said cellulosegenetic sequence possesses different catalytic activity and/orexpression characteristics, producing in turn modified cellulosedeposition in the target cell. In a particularly preferred embodiment ofthe invention, the endogenous cellulose gene of a plant is replaced witha gene which is merely capable of producing non-crystalline β-1,4-glucanpolymers or alternatively which is capable of producing a modifiedcellulose having properties similar to synthetic fibres such as rayon,in which the β-1,4-glucan polymers are arranged in an antiparallelconfiguration relative to one another.

The present invention extends to genetic constructs designed tofacilitate expression of a cellulose genetic sequence which isidentical, or complementary to the sequence set forth in any one or moreof SEQ ID Nos:1, 3, 4, 5, 7, 9, 11 or 13, or a functional derivative,part, homologue, or analogue thereof, or a genetic construct designed tofacilitate expression of a sense molecule, an antisense molecule,ribozyme molecule, co-suppression molecule, or gene targeting moleculecontaining said genetic sequence.

The said genetic construct of the present invention comprises theforegoing sense, antisense, or ribozyme, or co-suppression nucleic acidmolecule, or gene-targeting molecule, placed operably under the controlof a promoter sequence capable of regulating the expression of the saidnucleic acid molecule in a prokaryotic or eukaryotic cell, preferably aplant cell. The said genetic construct optionally comprises, in additionto a promoter and sense, or antisense, or ribozyme, or co-suppression,or gene-targeting nucleic acid molecule, a terminator sequence.

The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signals termination of transcription.Terminators are 3′-non-translated DNA sequences containing apolyadenylation signal, which facilitates the addition of polyadenylatesequences to the 3′-end of a primary transcript. Terminators active inplant cells are known and described in the literature. They may beisolated from bacteria, fungi, viruses, animals and/or plants. Examplesof terminators particularly suitable for use in the genetic constructsof the present invention include the nopaline synthase (NOS) geneterminator of Agrobacterium tumefaciens, the terminator of theCauliflower mosaic virus (CaMV) 35S gene, and the zein gene terminatorfrom Zea mays.

Reference herein to a “promoter” is to be taken in its broadest contextand includes the transcriptional regulatory sequences of a classicalgenomic gene, including the TATA box which is required for accuratetranscription initiation, with or without a CCAAT box sequence andadditional regulatory elements (i.e. upstream activating sequences,enhancers and silencers) which alter gene expression in response todevelopmental and/or external stimuli, or in a tissue-specific manner. Apromoter is usually, but not necessarily, positioned upstream or 5′, ofa structural gene, the expression of which it regulates. Furthermore,the regulatory elements comprising a promoter are usually positionedwithin 2 kb of the start site of transcription of the gene.

In the present context, the term “promoter” is also used to describe asynthetic or fusion molecule, or derivative which confers, activates orenhances expression of said sense, antisense, or ribozyme, orco-suppression nucleic acid molecule, in a plant cell. Preferredpromoters may contain additional copies of one or more specificregulatory elements, to further enhance expression of a sense antisense,ribozyme or co-suppression molecule and/or to alter the spatialexpression and/or temporal expression of said sense or antisense, orribozyme, or co-suppression, or gene-targeting molecule. For example,regulatory elements which confer copper inducibility may be placedadjacent to a heterologous promoter sequence driving expression of asense, or antisense, or ribozyme, or co-suppression, or gene-targetingmolecule, thereby conferring copper inducibility on the expression ofsaid molecule

Placing a sense or ribozyme, or antisense, or co-suppression, orgene-targeting molecule under the regulatory control of a promotersequence means positioning the said molecule such that expression iscontrolled by the promoter sequence. Promoters are generally positioned5′ (upstream) to the genes that they control. In the construction ofheterologous promoter/structural gene combinations it is generallypreferred to position the promoter at a distance from the genetranscription start site that is approximately the same as the distancebetween that promoter and the gene it controls in its natural setting,i.e., the gene from which the promoter is derived. As is known in theart, some variation in this distance can be accommodated without loss ofpromoter function. Similarly, the preferred positioning of a regulatorysequence element with respect to a heterologous gene to be placed underits control is defined by the positioning of the element in its naturalsetting, i.e., the genes from which it is derived. Again, as is known inthe art, some variation in this distance can also occur.

Examples of promoters suitable for use in genetic constructs of thepresent invention include viral, fungal, bacterial, animal and plantderived promoters capable of functioning in prokaryotic or eukaryoticcells. Preferred promoters are those capable of regulating theexpression of the subject cellulose genes of the invention in plantscells, fungal cells, insect cells, yeast cells, animal cells orbacterial cells, amongst others. Particularly preferred promoters arecapable of regulating expression of the subject nucleic acid moleculesin plant cells. The promoter may regulate the expression of the saidmolecule constitutively, or differentially with respect to the tissue inwhich expression occurs or, with respect to the developmental stage atwhich expression occurs, or in response to external stimuli such asphysiological stresses, or plant pathogens, or metal ions, amongstothers. Preferably, the promoter is capable of regulating expression ofa sense, or ribozyme, or antisense, or co-suppression molecule or genetargeting, in a plant cell. Examples of preferred promoters include theCaMV 35S promoter, NOS promoter, octopine synthase (OCS) promoter andthe like.

In a most preferred embodiment, the promoter is capable of expression inany plant cell, such as, but not limited to a plant selected from thelist comprising Arabidopsis thaliana, Oryza sativa, wheat, barley,maize, Brassica spp., Gossypium hirsutum and Eucalyptus spp., hemp,jute, flax, and woody plants including, but not limited to Pinus spp.,Populus spp., Picea spp., amongst others.

In a particularly preferred embodiment, the promoter may be derived froma genomic clone encoding a cellulose gene product, in particular thepromoter contained in the sequence set forth in SEQ ID NO:3 or SEQ IDNO:4. Preferably, the promoter sequence comprises nucleotide 1 to about1900 of SEQ ID NO:3 or nucleotides 1 to about 700 of SEQ ID NO:4 or ahomologue, analogue or derivative capable of hybridizing thereto underat least low stringency conditions.

Optionally, the genetic construct of the present invention furthercomprises a terminator sequence.

In an exemplification of this embodiment, there is provided a binarygenetic construct comprising the isolated nucleotide sequence ofnucleotides set forth in SEQ ID NO:3. There is also provided a geneticconstruct comprising the isolated nucleotide sequence of nucleotides setforth in SEQ ID NO:1, in the antisense orientation, placed operably inconnection with the CaMV 35S promoter.

In the present context, the term “in operable connection with” meansthat expression of the isolated nucleotide sequence is under the controlof the promoter sequence with which it is connected, regardless of therelative physical distance of the sequences from each other or theirrelative orientation with respect to each other.

An alternative embodiment of the invention is directed to a geneticconstruct comprising a promoter or functional derivative, part,fragment, homologue, or analogue thereof, which is capable of directingthe expression of a polypeptide early in the development of a plant cellat a stage when the cell wall is developing, such as during cellexpansion or during cell division. In a particularly preferredembodiment, the promoter is contained in the sequence set forth in SEQID NO:3 or SEQ ID NO:4. Preferably, the promoter sequence comprisesnucleotide 1 to about 1900 of SEQ ID NO:3 or nucleotides 1 to about 700of SEQ ID NO:4 or a homologue, analogue or derivative capable ofhybridizing thereto under at least low stringency conditions.

The polypeptide may be a reporter molecule which is encoded by a genesuch as the bacterial β-glucuronidase gene or chloramphenicolacetyltransferase gene or alternatively, the firefly luciferase gene.Alternatively, the polypeptide may be encoded by a gene which is capableof producing a modified cellulose in the plant cell when placed incombination with the normal complement of cellulose genes which areexpressible therein, for example it may be a cellulose-like geneobtained from a bacterial or fungal source or a cellulose gene obtainedfrom a plant source.

The genetic constructs of the present invention are particularly usefulin the production of crop plants with altered cellulose content orstructure. In particular, the rate of cellulose deposition may bereduced leading to a reduction in the total cellulose content of plantsby transferring one or more of the antisense, ribozyme or co-suppressionmolecules described supra into a plant or alternatively, the same orsimilar end-result may be achieved by replacing an endogenous cellulosegene with an inactive or modified cellulose gene using gene-targetingapproaches. The benefits to be derived from reducing cellulose contentin plants are especially apparent in food and fodder crops such as, butnot limited to maize, wheat, barley, rye, rice, barley, millet orsorghum, amongst others where improved digestibility of said crop isdesired. The foregoing antisense, ribozyme or co-suppression moleculesare also useful in producing plants with altered carbon partitioningsuch that increased carbon is available for growth, rather thandeposited in the form of cellulose.

Alternatively, the introduction to plants of additional copies of acellulose gene in the ‘sense’ orientation and under the control of astrong promoter is useful for the production of plants with increasedcellulose content or more rapid rates of cellulose biosynthesis.Accordingly, such plants may exhibit a range of desired traitsincluding, but not limited to modified strength and/or shape and/orproperties of fibres, cell and plants, increased protection againstchemical, physical or environmental stresses such as dehydration, heavymetals (e.g. cadmium) cold, heat or wind, increased resistance to attackby pathogens such as insects, nematodes and the like which physicallypenetrate the cell wall barrier during invasion/infection of the plant.

Alternatively, the production of plants with altered physical propertiesis made possible by the introduction thereto of altered cellulosegene(s). Such plants may produce β-1,4-glucan which is eithernon-crystalline or shows altered crystallinity. Such plants may alsoexhibit a range of desired traits including but not limited to, altereddietary fibre content, altered digestibility and degradability orproducing plants with altered extractability properties.

Furthermore, genetic constructs comprising a plant cellulose gene in the‘sense’ orientation may be used to complement the existing range ofcellulose genes present in a plant, thereby altering the composition ortiming of deposition of cellulose deposited in the cell wall of saidplant. In a preferred embodiment, the cellulose gene from one plantspecies or a β-1,4-glucan synthase gene from a non-plant species is usedto transform a plant of a different species, thereby introducing novelcellulose biosynthetic metabolism to the second-mentioned plant species.

In a related embodiment, a recombinant fusion polypeptide may beproduced containing the active site from one cellulose gene productfused to another cellulose gene product, wherein said fusion polypeptideexhibits novel catalytic properties compared to either ‘parent’polypeptide from which it is derived. Such fusion polypeptides may beproduced by conventional recombinant DNA techniques known to thoseskilled in the art, either by introducing a recombinant DNA capable ofexpressing the entire fusion polypeptide into said plant oralternatively, by a gene-targeting approach in which recombination atthe DNA level occurs in vivo and the resultant gene is capable ofexpressing a recombinant fusion polypeptide.

The present invention extends to all transgenic methods and productsdescribed supra, including genetic constructs.

The recombinant DNA molecule carrying the sense, antisense, ribozyme orco-suppression molecule of the present invention and/or geneticconstruct comprising the same, may be introduced into plant tissue,thereby producing a “transgenic plant”, by various techniques known tothose skilled in the art. The technique used for a given plant speciesor specific type of plant tissue depends on the known successfultechniques. Means for introducing recombinant DNA into plant tissueinclude, but are not limited to, transformation (Paszkowski et al.,1984), electroporation (Fromm et al., 1985), or microinjection of theDNA (Crossway et al., 1986), or T-DNA-mediated transfer fromAgrobacterium to the plant tissue. Representative T-DNA vector systemsare described in the following references: An et al. (1985);Herrera-Estrella et al. (1983a,b); Herrera-Estrella et al. (1985). Onceintroduced into the plant tissue, the expression of the introduced genemay be assayed in a transient expression system, or it may be determinedafter selection for stable integration within the plant genome.Techniques are known for the in vitro culture of plant tissue, and in anumber of cases, for regeneration into whole plants. Procedures fortransferring the introduced gene from the originally transformed plantinto commercially useful cultivars are known to those skilled in theart.

A still further aspect of the present invention extends to a transgenicplant such as a crop plant, carrying the foregoing sense, antisense,ribozyme, co-suppression, or gene-targeting molecule and/or geneticconstructs comprising the same. Preferably, the transgenic plant is oneor more of the following: Arabidopsis thaliana, Oryza sativa, wheat,barley, maize, Brassica spp., Gossypium hirsutum and Eucalyptus spp.,hemp, jute, flax, Pinus spp., Populus spp., or Picea spp. Additionalspecies are not excluded.

The present invention further extends to the progeny of said transgenicplant.

Yet another aspect of the present invention provides for the expressionof the subject genetic sequence in a suitable host (e.g. a prokaryote oreukaryote) to produce full length or non-full length recombinantcellulose gene products.

Hereinafter the term “cellulose gene product” shall be taken to refer toa recombinant product of a cellulose gene as hereinbefore defined.Accordingly, the term “cellulose gene product” includes a polypeptideproduct of any gene involved in the cellulose biosynthetic pathway inplants, such as, but not limited to a cellulose synthase gene product.

Preferably, the recombinant cellulose gene product comprises an aminoacid sequence having the catalytic activity of a cellulose synthasepolypeptide or a functional mutant, derivative part, fragment, oranalogue thereof.

In a particularly preferred embodiment of the invention, the recombinantcellulose gene product comprises a sequence or amino acids that is atleast 40% identical to any one or more of SEQ ID Nos:2, 6, 8, 10, 12 or14, or a homologue, analogue or derivative thereof.

Single and three-letter abbreviations used for amino acid residuescontained in the specification are provided in Table 1.

In the present context, “homologues” of an amino acid sequence refer tothose polypeptides, enzymes or proteins which have a similar catalyticactivity to the amino acid sequences described herein, notwithstandingany amino acid substitutions, additions or deletions thereto. Ahomologue may be isolated or derived from the same or another plantspecies as the species from which the polypeptides of the invention arederived.

“Analogues” encompass polypeptides of the invention notwithstanding theoccurrence of any non-naturally occurring amino acid analogues therein.

“Derivatives” include modified peptides in which ligands are attached toone or more of the amino acid residues contained therein, such ascarbohydrates, enzymes, proteins, polypeptides or reporter moleculessuch as radionuclides or fluorescent compounds. Glycosylated,fluorescent, acylated or alkylated forms of the subject peptides areparticularly contemplated by the present invention. Additionally,derivatives of an amino acid sequence described herein which comprisesfragments or parts of the subject amino acid sequences are within thescope of the invention, as are homopolymers or heteropolymers comprisingtwo or more copies of the subject polypeptides. Procedures forderivatizing peptides are well-known in the art.

TABLE 1 Three-letter One-letter Amino Acid Abbreviation Symbol AlanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CD-alanine Dal X Glutamine Gln Q Glutamic acid Glu E Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tryosine Tyr Y Valine Val V Any amino acid Xaa X

Substitutions encompass amino acid alterations in which an amino acid isreplaced with a different naturally-occurring or a non-conventionalamino acid residue. Such substitutions may be classified as“conservative”, in which an amino acid residue contained in a cellulosegene product is replaced with another naturally-occurring amino acid ofsimilar character, for example Gly⇄Ala, Val⇄Ile⇄Leu, Asp⇄Glu, Lys⇄Arg,Asn⇄Gln or Phe⇄Trp⇄Tyr.

Substitutions encompassed by the present invention may also be“non-conservative”, in which an amino acid residue which is present in acellulose gene product described herein is substituted with an aminoacid with different properties, such as a naturally-occurring amino acidfrom a different group (e.g. substituted a charged or hydrophobic aminoacid with alanine), or alternatively, in which a naturally-occurringamino acid is substituted with a non-conventional amino acid.

Non-conventional amino acids encompassed by the invention include, butare not limited to those listed in Table 2.

Amino acid substitutions are typically of single residues, but may be ofmultiple residues, either clustered or dispersed.

Amino acid deletions will usually be of the order of about 1–10 aminoacid residues, while insertions may be of any length. Deletions andinsertions may be made to the N-terminus, the C-terminus or be internaldeletions or insertions. Generally, insertions within the amino acidsequence will be smaller than amino- or carboxy-terminal fusions and ofthe order of 1–4 amino acid residues.

A homologue, analogue or derivative of a cellulose gene product asreferred to herein may readily be made using peptide synthetictechniques well-known in the art, such as solid phase peptide synthesisand the like, or by recombinant DNA manipulations. Techniques for makingsubstituent mutations at pre-determined sites using recombinant DNAtechnology, for example by M13 mutagenesis, are also well-known. Themanipulation of nucleic acid molecules to produce variant peptides,polypeptides or proteins which manifest as substitutions, insertions ordeletions are well-known in the art.

The cellulose gene products described herein may be derivatized furtherby the inclusion or attachment thereto of a protective group whichprevents, inhibits or slows proteolytic or cellular degradativeprocesses. Such derivatization may be useful where the half-life of thesubject polypeptide is required to be extended, for ample to increasethe amount of cellulose produced in a primary or secondary cell wall ofa plant cell or alternatively, to increase the amount of proteinproduced in a bacterial or eukaryotic expression system. Examples ofchemical groups suitable for this purpose include, but are not limitedto, any of the non-conventional amino acid residues listed in Table 2,in particular a D-stereoisomer or a methylated form of anaturally-occurring amino acid listed in Table 1. Additional chemicalgroups which are useful for this purpose are selected from the listcomprising aryl or heterocyclic N-acyl substituents, polyalkylene oxidemoieties, desulphatohirudin muteins, alpha-muteins,alpha-aminophosphonic acids, water-soluble polymer groups such aspolyethylene glycol attached to sugar residues using hydrazone or oximegroups, benzodiazepine dione derivatives, glycosyl groups such asbeta-glycosylamine or a derivative thereof, isocyanate conjugated to apolyol functional group or polyoxyethylene polyol capped withdiisocyanate, amongst others. Similarly, a cellulose gene product or ahomologue, analogue or derivative thereof may be cross-linked or fusedto itself or to a protease inhibitor peptide, to reduce susceptibilityof said molecule to proteolysis.

TABLE 2 Non-conventional Non-conventional amino acid Code amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine NmhpheN-(N-(2,2-diphenylethyl)carbamylmethyl) NnbhmN-(N-(3,3-diphenylpropyl)carbamylmethyl) Nnbhe glycine glycine1-carboxy-1-(2,2-diphenyl-ethylamino) Nmbc cyclopropane

In an alternative embodiment of the invention, the recombinant cellulosegene product is characterised by at least one functional β-glycosyltransferase domain contained therein.

The term “β-glycosyl transferase domain” as used herein refers to asequence of amino acids which is highly conserved in differentprocessive enzymes belonging to the class of glycosyl transferaseenzymes (Saxena et al., 1995), for example the bacterial 1-1,4-glycosyltransferase enzymes and plant cellulose synthase enzymes amongst others,wherein said domain possesses a putative function in contributing to ormaintaining the overall catalytic activity, substrate specificity orsubstrate binding of an enzyme in said enzyme class. The β-glycosyltransferase domain is recognisable by the occurrence of certain aminoacid residues at particular locations in a polypeptide sequence, howeverthere is no stretch of contiguous amino acid residues comprised therein.

As a consequence of the lack of contiguity in a β-glycosyl transferasedomain, it is not a straightforward matter to isolate a cellulose geneby taking advantage of the presence of a β-glycosyl transferase domainin the polypeptide encoded by said gene. For example, the β-glycosyltransferase domain would not be easily utilisable as a probe tofacilitate the rapid isolation of all β-glycosyl transferase geneticsequences from a particular organism and then to isolate from thosegenetic sequences a cellulose gene such as cellulose synthase.

In a preferred embodiment, the present invention provides an isolatedpolypeptide which:

-   -   (i) contains at least one structural 1-glycosyl transferase        domain as hereinbefore defined; and    -   (ii) has at least 40% amino acid sequence similarity to at least        20 contiguous amino acid residues set forth in any one or more        of SEQ ID Nos:2, 6, 8, 10, 12 or 14, or a homologue, analogue or        derivative thereof.

More preferably, the polypeptide of the invention is at least 40%identical to at least 50 contiguous amino acid residues, even morepreferably at least 100 amino acid residues of any one or more of SEQ IDNos:2, 6, 8, 10, 12 or 14, or a homologue, analogue or derivativethereof.

In a particularly preferred embodiment, the percentage similarity to anyone or more of SEQ ID Nos:2, 6, 8, 10, 12 or 14 is at least 50–60%, morepreferably at least 65–70%, even more preferably at least 75–80% andeven more preferably at least 85–90%, including about 91% or 95%.

In a related embodiment, the present invention provides a “sequencablypure” form of the amino acid sequence described herein. “Sequencablypure” is hereinbefore described as substantially homogeneous tofacilitate amino acid determination.

In a further related embodiment, the present invention provides a“substantially homogeneous” form of the subject amino acid sequence,wherein the term “substantially homogeneous” is hereinbefore defined asbeing in a form suitable for interaction with an immunologicallyinteractive molecule. Preferably, the polypeptide is at least 20%homogeneous, more preferably at least 50% homogeneous, still morepreferably at least 75% homogeneous and yet still more preferably atleast about 95–100% homogenous, in terms of activity per microgram oftotal protein in the protein preparation.

The present invention further extends to a synthetic peptide of at least5 amino acid residues in length derived from or comprising a part of theamino acid sequence set forth in any one or more of SEQ ID Nos:2, 6, 8,10, 12 or 14, or having at least 40% similarity thereto.

Those skilled in the art will be aware that such synthetic peptides maybe useful in the production of immunologically interactive molecules forthe preparation of antibodies or as the peptide component of animmunoassay.

The invention further extends to an antibody molecule such as apolyclonal or monoclonal antibody or an immunologically interactive partor fragment thereof which is capable of binding to a cellulose geneproduct according to any of the foregoing embodiments.

The term “antibody” as used herein, is intended to include fragmentsthereof which are also specifically reactive with a polypeptide of theinvention. Antibodies can be fragmented using conventional techniquesand the fragments screened for utility in the same manner as for wholeantibodies. For example, F(ab′)₂ fragments can be generated by treatingantibody with pepsin. The resulting F(ab′)₂ fragment can be treated toreduce disulfide bridges to produce Fab′ fragments.

Those skilled in the art will be aware of how to produce antibodymolecules when provided with the cellulose gene product of the presentinvention. For example, by using a polypeptide of the present inventionpolyclonal antisera or monoclonal antibodies can be made using standardmethods. A mammal, (e.g., a mouse, hamster, or rabbit) can be immunizedwith an immunogenic form of the polypeptide which elicits an antibodyresponse in the mammal. Techniques for conferring immunogenicity on apolypeptide include conjugation to carriers or other techniques wellknown in the art. For example, the polypeptide can be administered inthe presence of adjuvant. The progress of immunization can be monitoredby detection of antibody titers in plasma or serum. Standard ELISA orother immunoassay can be used with the immunogen as antigen to assessthe levels of antibodies. Following immunization, antisera can beobtained and, if desired IgG molecules corresponding to the polyclonalantibodies may be isolated from the sera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused with myeloma cellsby standard somatic cell fusion procedures thus immortalizing thesecells and yielding hybridoma cells. Such techniques are well known inthe art. For example, the hybridoma technique originally developed byKohler and Milstein (1975) as well as other techniques such as the humanB-cell hybridoma technique (Kozbor et al., 1983), the EBV-hybridomatechnique to produce human monoclonal antibodies (Cole et al., 1985),and screening of combinatorial antibody libraries (Huse et al., 1989).Hybridoma cells can be screened immunochemically for production ofantibodies which are specifically reactive with the polypeptide andmonoclonal antibodies isolated.

As with all immunogenic compositions for eliciting antibodies, theimmunogenically effective amounts of the polypeptides of the inventionmust be determined empirically. Factors to be considered include theimmunogenicity of the native polypeptide, whether or not the polypeptidewill be complexed with or covalently attached to an adjuvant or carrierprotein or other carrier and route of administration for thecomposition, i.e. intravenous, intramuscular, subcutaneous, etc., andthe number of immunizing doses to be administered. Such factors areknown in the vaccine art and it is well within the skill ofimmunologists to make such determinations without undue experimentation.

It is within the scope of this invention to include any secondantibodies (monoclonal, polyclonal or fragments of antibodies) directedto the first mentioned antibodies discussed above. Both the first andsecond antibodies may be used in detection assays or a first antibodymay be used with a commercially available anti-immunoglobulin antibody.

The present invention is further described by reference to the followingnon-limiting Figures and Examples.

In the Figures:

FIG. 1 is a photographic representation showing the inflorescence lengthof wild-type Arabidopsis thaliana Columbia plants (plants 1 and 3) andrsw1 plants (plants 2 and 4) grown at 21° C. (plants 1 and 2) or 31° C.Plants were grown initially at 21° C. until bolting commenced, the boltswere removed and the re-growth followed in plants grown at eachtemperature.

FIG. 2 is a photographic representation of a cryo-scanning electronmicrograph showing misshapen epidermal cells in the cotyledons andhypocotyl of the rsw1 mutant when grown at 31° C. for 10 days.

FIG. 3 is a graphical reprsentation of a gas chromatograph of alditolacetates of methylated sugars from a cellulose standard (top panel) andfrom the neutral glucan derived from shoots of rsw1 plants grown at 31°C. (lower panel). The co-incident peaks show that the rsw1 glucan is1,4-linked.

FIG. 4 is a schematic representation of the contiguous region ofArabidopsis thaliana chromosome 4 (stippled box) between the cosmidmarkers g8300 and 06455, showing the location of overlapping YAC clones(open boxes) within the contiguous region. The position of the RSW1locus is also indicated, approximately 1.2cM from g8300 and 0.9cM from06455. The scale indicates 100 kb in length. L, left-end of YAC; R,right-end of YAC. Above the representation of chromosome 4, the YACfragments and cosmid clone fragments used to construct the contiguousregion are indicated, using a prefix designation corresponding to theYAC or cosmid from which the fragments were obtained (e.g. yUP9E3,yUP20B12, etc) and a suffix designation indicating whether the fragmentcorresponds to the right-end (RE) or left-end (LE) of the YAC clone; N,North; S, South; CAPS, cleaved amplified polymorphic sequence (Koniecznyand Ausubel, 1993) version of the g8300 marker.

FIG. 5 is a schematic representation of a restriction map of construct23H 12 between the left T-DNA border (LB) and right T-DNA border (RB)sequences (top solid line), showing the position of the Arabidopsisthaliana RSW1 locus (stippled box). The line at the top of the figureindicates the region of 23H12 which is contained in construct pRSW1. Thestructure of the RSW1 gene between the translation start (ATG) andtranslation stop (TAG) codons is indicated at the bottom of the figure.Exons are indicated by filled boxes; introns are indicated by the solidblack line. The alignment of EST clone T20782 to the 3′-end of the RSW1gene, from near the end of exon 7 to the end of exon 14, is alsoindicated at the bottom of the figure. Restriction sites within 23H 12are as follows: B, BamHI; E, EcoRI; H, HindIII; S, SalI; Sm, Smal.

FIG. 6 is a photographic representation showing complementation of theradial root swelling phenotype of the rsw1 mutant by transformation withconstruct 23H12. The rsw1 mutant was transformed with 23H12 as describedin Example 6. Transformed rsw1 plants (centre group of three seedlings),untransformed rsw1 plants (left group of three seedlings) anduntransformed A. thaliana Columbia plants (right group of threeseedlings) were grown at 21° C. for 5 days and then transferred to 31°C. for a further 2 days, after which time the degree of root elongationand radial root swelling was determined.

FIG. 7 is a photographic representation comparing wild-type Arabidopsisthaliana Columbia plants (right-hand side of the ruler) and A. thalianaColumbia plants transformed with the antisense RSW1 construct (i.e. ESTT20782 expressed in the antisense orientation under control of the CaMV35S promoter sequence; left-hand side of the ruler), showinginflorescence shortening at 21° C. in plants transformed with theantisense RSW1 construct compared to untransformed Columbia plants. Thephenotype of the antisense plants at 21° C. is similar to the phenotypeof the rsw1 mutant at 31° C. Inflorescence height is indicated inmillimeters.

FIG. 8 is a schematic representation showing the first 90 amino acidresidues of Arabidopsis thaliana RSW1 aligned to the amino acidsequences of homologous polypeptides from A. thaliana and other plantspecies. The shaded region indicates highly conserved sequences. Ath-Aand Ath-B are closely related Arabidopsis thaliana cDNA clonesidentified by hybridisation screening using part of the RSW1 cDNA as aprobe. SO₅₄₂, rice EST clone (MAFF DNA bank, Japan); celA1 and celA2,cotton cDNA sequences expressed in cotton fibre (Pear et al. 1996);SOYSTF1A and SOYSTF1B, putative soybean bZIP transcription factors.Amino acid designations are as indicated in Table 1 incorporated herein.Conserved cysteine residues are indicated by the asterisk.

FIGS. 9A–9J are schematic representations showing the alignment of thecomplete amino acid sequence of Arabidopsis thaliana RSW1 to the aminoacid sequences of homologous polypeptides from A. thaliana and otherplant species. The shaded region indicates highly conserved sequences.Ath-A and Ath-B are closely related Arabidopsis thaliana cDNA clonesidentified by hybridisation screening using part of the RSW1 cDNA as aprobe. SO₅₄₂, rice EST clone (MAFF DNA bank, Japan); celA1, cottongenetic sequence (Pear et al. 1996); D48636, a partial cDNA cloneobtained from rice (Pear et al. 1996). Amino acid designations are asindicated in Table 1 incorporated herein. Numbering indicates the aminoacid position in the RSW1 sequence.

FIG. 10 is a schematic representation of the RSW1 polypeptide, showingthe positions of putative transmembrane helices (hatched boxes),cysteine-rich region (Cys) and aspartate residues (D) and the QVLRWsignature which are conserved between RSW1 and related amino acidsequences. Regions of RSW1 which are highly-conserved between putativecellulose biosynthesis polypeptides are indicated by the dark-shadedboxes, while less-conserved regions are indicated by the light-shadedboxes.

FIG. 11 is a photographic representation of a Southern blothybridisation of the 5′-end of the Arabidopsis thaliana RSW1 cDNA toBglII-digested DNA derived from A. thaliana (lane 1) and cotton (lane2). Hybridisations were carried out under low stringency conditions at55° C. Arrows indicate the positions of hybridising bands.

EXAMPLE 1 Characterisation of the Cellulose-Deficient Arabidopsisthaliana Mutant rsw1

1. Morphology

The Arabidopsis thaliana rsw1 mutant was produced in a geneticbackground comprising the ecotype Columbia.

The altered root cell-shape and temperature sensitivity of the rootmorphology of the Arabidopsis thaliana mutant rsw1 are disclosed, amongother morphological mutants, by Baskin et al. (1992).

As shown in FIG. 1, the present inventors have shown that the rsw1mutant exhibits the surprising phenotype of having reduced inflorescenceheight when grown at 31° C., compared to wild-type Columbia plants grownunder similar conditions. In contrast, when grown at 21° C., theinflorescence height of rsw1 is not significantly different from wildtype plants grown under similar conditions, indicating that the shootphenotype of rsw1 is conditional and temperature-dependent.

Furthermore, cryo-scanning electron microscopy of the epidermal cells ofthe rsw1 mutant indicates significant abnormality in cell shape,particularly in respect of those epidermal cells forming the leaves,hypocotyl and cotyledons, when the seedlings are grown at 31° C. (FIG.2).

Rosettes (terminal complexes) are the putative hexameric cellulosesynthase complexes of higher plant plasma membranes (Herth, 1985).Freeze-fractured root cells of Arabidopsis thaliana rsw1 plants grown at18° C. show cellulose microfibrils and rosettes on the PF face of theplasma membrane that resembles those of wild-type A. thaliana and otherangiosperms. Transferring the rsw1 mutant to 31° C. reduces the numberof rosettes in the mutant within 30 min, leading to extensive loss after3 hours. Plasma membrane particles align in rows on prolonged exposureto the restrictive temperature. In contrast, there is no change in theappearance of cortical microtubules that align cellulose microfibrils,or of Golgi bodies that synthesise other wall polysaccharides andassemble rosettes.

2. Carbohydrate Content

The effect of mutations in the RSW1 gene on the synthesis of celluloseand other carbohydrates was assessed by measuring in vivo incorporationof ¹⁴C (supplied as uniformly labeled glucose) into various cell wallfractions. Wild type (RSW1) and homozygous mutant rsw1 seed weregerminated at 21° C. on agar containing Hoagland's nutrients and 1%(w/v) unlabelled glucose. After 5 d, half of the seedlings weretransferred to 31° C. for 1 d while the remainder was maintained at 21°C. for the same time. Seedlings were covered with a solution containingHoagland's nutrients and ¹⁴C-glucose and incubated for a further 3 h atthe same temperature. Rinsed roots and shoots were separated and frozenin liquid nitrogen. Tissue was homogenised in cold, 0.5 M potassiumphosphate buffer (0.5M KH₂PO₄, pH 7.0) and a crude cell wall fractioncollected by centrifugation at 2800 rpm. The wall fraction was extractedwith chloroform/methanol [1:1 (v/v)] at 40° C. for 1 hour, followed by abrief incubation at 150° C., to remove lipids. The pellet was washedsuccessively with 2 ml methanol, 2 ml acetone and twice with 2 ml ofdeionised water. Finally, the pellet was extracted successively withdimethyl sulphoxide under nitrogen to remove starch; 0.5% ammoniumoxalate to remove pectins; 0.1 M KOH and 3 mg/ml NaBH₄ and then with 4 MKOH and 3 mg/ml NaBH₄ to extract hemicelluloses; boiling aceticacid/nitric acid/water [8:1:2 (v/v)], to extract any residualnon-cellulosic carbohydrates and leave crystalline cellulose as thefinal insoluble pellet (Updegraph, 1969). All fractions were analysed byliquid scintillation counting and the counts in each fraction from themutant were expressed as a percentage of the counts in the wild typeunder the same conditions.

As shown in Table 3, mutant and wild type plants behave in quite similarfashion at 21° C. (the permissive temperature) whereas, at therestrictive temperature of 31° C., the incorporation of ¹⁴C intocellulose is severely inhibited (to 36% of wild type) by the rsw1mutation. The data in Table 3 indicate that cellulose synthesis isspecifically inhibited in the rsw1 mutant. The wild type RSW1 gene istherefore involved quite directly in cellulose synthesis and changingits sequence by mutation changes the rate of synthesis.

TABLE 3 Counts in fractions from rsw1 plants expressed as a % of countsin comparable fraction from wild type plants Pectins HemicellulosesCellulose 21° C. 31° C. 21° C. 31° C. 21° C. 31° C. 125 104 111 101 8036

In homozygous mutant rsw1 plants, the pectin fraction extracted byammonium oxalate contained abundant glucose, a typical of true uronicacid-rich pectins. The great majority of the glucose remained in thesupernatant when cetyltrimethylammonium bromide precipitated thenegatively charged pectins.

3. Non-crystalline β-1,4-glucan content

The quantity of cellulose and the quantity of a non-crystallineβ-1,4-glucan recovered from the ammonium oxalate fraction weredetermined for seedlings of wild type Columbia and for backcrossed,homozygous rsw1 that were grown for either 7 days at 21° C. oralternatively, for 2 days at 21° C. and 5 days at 31° C., on verticalagar plates containing growth medium (Baskin et al., 1992) plus 1% (w/v)glucose, and under continuous light (90 μmol m⁻² s⁻¹). Roots and shootswere separated from about 150 seedlings, freeze-dried to constant weightand ground in a mortar and pestle with 3 ml of cold 0.5 M potassiumphosphate buffer (pH 7.0). The combined homogenate after two bufferrinses (2 ml each) was centrifuged at 2800×g for 10 min. After washingthe pellet fraction twice with 2 ml buffer and twice with 2 ml distilledwater, the pellet, comprising the crude cell wall fraction, and thepooled supernatants, comprising the phosphate buffer fraction wereretained. The crude cell wall pellet fraction was stirred with two 3 mlaliquots of chloroform/methanol [1:1 (v/v)] for 1 hour at 40° C., 2 mlof methanol at 40° C. for 30 min, 2 ml of acetone for 30 min, and twicewith water. The whole procedure repeated in the case of shoots. Combinedsupernatants were dried in a nitrogen stream. The pellet wassuccessively extracted with: (i) 3 ml of DMSO-water 9:1 [v/v], sealedunder nitrogen, overnight with shaking, followed by two 2 ml extractionsusing DMSO/water and three 2 ml water washes; (ii) 3 ml of ammoniumoxalate (0.5%) at 100° C. for 1 hour, followed by two water washes;(iii) 3 ml of 0.1 M KOH containing 1 mg/ml sodium borohydride, for 1hour at 25° C. (repeated once for root material or twice for shootmaterial), with a final wash with 2 ml water; (iv) 3 ml of 4 M KOHcontaining 1 mg/ml sodium borohydride, for 1 hour at 25° C. (repeatedonce for root material or twice for shoot material). The final pelletwas boiled with intermittent stirring in 3 ml of acetic acid-nitricacid-water [8:1:2 (v/v)] (Updegraph 1969), combined with 2 water washes,and diluted with 5 ml water.

The insoluble residue of cellulose was solubilised in 67% (v/v) H₂SO₄,shown to contain greater than 97% (w/v) glucose using GC/MS (FisonsAS800/MD800) of alditol acetates (Doares et al., 1991) and quantified inthree independent samples by anthrone/H₂SO₄ reaction. Results of GC/MSfor pooled replica samples are presented in Table 4.

The non-crystalline β-1,4-glucan was recovered as the supernatant fromthe ammonium oxalate fraction when anionic pectins were precipitated byovernight incubation at 37° C. with 2% (w/v) cetyltrimethylammoniumbromide (CTAB) and collected by centrifugation at 2800×g for 10 min. Theglucan (250 μg/ml) or starch (Sigma; 200 μg/ml) were digested withmixtures of endocellulase (EC 3.2.1.4; Megazyme, Australia) fromTrichoderma and almond β-glucosidase (EC 3.2.1.21; Sigma), or Bacillussp. α-amylase (EC 3.2.1.1; Sigma) and rice α-glucosidase (EC 3.2.1.20;Sigma).

The material recovered in the supernatant from the ammonium oxalatefraction was shown to contain a pure β-1,4-glucan by demonstrating that:

-   -   (i) only glucose was detectable when it was hydrolysed by 2 M        TFA in a sealed tube for 1 h at 120° C. in an autoclave, the        supernatant (2000 g for 5 min) was dried under vacuum at 45° C.        to remove TFA and glucose was determined by GC/MS;    -   (ii) methylation (Needs and Selvendran 1993) gave a dominant        peak resolved by thin layer chromatography and by GC/MS that was        identical to that from a cellulose standard and so indicative of        1,4-linked glucan (FIG. 3); and

(iii) the endo-cellulase and β-1,4-glucosidase mixture released 83% ofthe TFA-releasable glucose from the glucan produced by rsw1 at 31° C.while the α-amylase/α-glucosidase mixture released no glucose from theglucan. Conversely, the α-amylase/β-glucosidase mixture released 95% ofthe TFA-releasable glucose from a starch sample, while theendo-cellulase/β-1,4-glucosidase mixture released no glucose fromstarch.

Extractability of the glucan using ammonium oxalate, and thesusceptibility of the glucan to endocellulase/β-glucosidase and TFAhydrolysis indicate that the glucan in the rsw1 mutant is notcrystalline, because it is the crystallinity of glucan which makescellulose resistant to extraction and degradation.

Table 4 shows the quantity of glucose in cellulose determined by theanthrone/H₂SO₄ reaction and the quantity in the non-crystalline glucanafter TFA hydrolysis, for shoots of wild type and mutant rsw1Arabidopsis plants. The data indicate that the production of celluloseand of the non-crystalline β-1,4-glucan can be manipulated by mutationalchanges in the RSW1 gene.

TABLE 4 Glucose contents of cellulose and of the ammoniumoxalate-extractable glucan wild type rsw1 21° C. 31° C. 21° C. 31° C.Cellulose 273 + 28 363 + 18* 218 + 20 159 + 19* Glucan 22 58 24 195 Allvalues nmol glucose mg-1 plant dry weight + sd (n = 3). *Differencessignificant at 0.001% level.4. Starch Content

The quantity of starch recovered in the DMSO fraction from roots in theexperiment described above was also determined by the anthrone/H₂SO₄extraction (Table 5).

As shown in Table 5, the level of starch deposited in the rsw1 mutant is4-fold that detectable in the roots of wild-type plants at therestrictive temperature of 31° C. A similar rise in starch is also seenif the data are expressed as nmol glucose per plant. There is nodetectable difference in deposition at starch between rsw1 plants andwild-type plants at 21° C.

TABLE 5 Quantity of starch (nmol glucose per mg dry weight of seedling)extracted from roots of rsw1 and wild type seedlings PhenotypeTemperature Wild-type rsw1 mutant 21° C. 22 18 31° C. 37 126

The composition of cell walls in the rsw1 mutant plant compared to wildtype plants at the restrictive temperature of 31° C., is summarised inTable 6.

TABLE 6 Mol % composition of cell walls from shoots of rsw1 andwild-type seedlings grown at 31° C. Phenotype Cell wall componentWild-type rsw1 mutant Crystalline cellulose 38.4 16.5 Non-crystalline8.5 27.1 β-1,4-glucan Pectin 37.1 36.3 Alkali-soluble 15.6 19.8Acid-soluble 0.3 0.4

In conclusion, the rsw1 mutation disassembles cellulose synthasecomplexes in the plasma membrane, reduces cellulose accumulation andcauses β-1,4-glucan to accumulate in a non-crystalline form.

EXAMPLE 2 Mapping of YAC Clones to the rsw1 Locus

The rsw1 locus in the mutant Arabidopsis thaliana plant described inExample 1 above was mapped to chromosome 4 of A. thaliana using RFLPgene mapping techniques (Chang et al., 1988; Nam et al., 1989) toanalyse the F₂ or F₃ progeny derived from a Columbia (Co)/Landsberg(Ler) cross. In particular, the rsw1 mutation was shown to be linkedgenetically to the ga5 locus, which is a chromosome 4 visual marker inA. thaliana.

Based on an analysis of map distances and chromosomal break points in293 F₂ or F₃ progeny derived from a Columbia (Co)/Landsberg (Ler) cross,rsw1 was localised to an approximately 2.1 cM region between the RFLPmarkers g8300 and 06455, approximately 1.2cM south of the CAPS (cleavedamplified polymorphic sequence; Konieczny and Ausubel, 1993) version ofthe g8300 marker (FIG. 4).

The interval between g8300 and 06455 in which rsw1 residues was found tobe spanned by an overlapping set of Yeast Artificial Chromosome (YAC)clones. The clones were obtained from Plant Industry, CommonwealthScientific and Industrial Research Organisation, Canberra, Australia.The YACs were positioned in the g8300/06455 interval by hybridisationusing known DNA molecular markers (from within the interval) and DNAfragments from the ends of the YACs. The length of the interval wasestimated to comprise 900 kb of DNA.

Refined gene mapping of recombinants within the region spanned by YACclones established the genetic distance between the RFLP marker g8300and the rsw1 locus.

The combination of genetic map distance data and the mapping of YACclones within the region further localised the rsw1 locus to the YACclone designated yUP5C8.

EXAMPLE 3 Mapping of cDNA Clones to the YAC Clone YUP5C8

An Arabidopsis thaliana cDNA clone designated T20782 was obtained fromthe public Arabidopsis Resource Centre, Ohio State University, 1735 NeilAvenue, Columbus, Ohio 43210, United States of America. The T20782 cDNAclone was localised broadly to the DNA interval on Arabidopsischromosome 4 between the two markers g8300 and 06455 shown in FIG. 4.Using a polymerase chain reaction (PCR) based approach DNA primers(5′-AGAACAGCAGATACACGGA-3′ SEQ ID NO:15 and 5′-CTGAAGAAGGCTGGACAAT-3′,SEQ ID NO:16) designed to the T20782 cDNA nucleotide sequence were usedto screen Arabidopsis YAC clone libraries. The T20782 cDNA clone wasfound to localise to YACs (CIC1F9, CIC10E9, CIC11D9) identified on theArabidopsis chromosome 4 g8300 and 06455 interval (FIG. 4). The sameapproach was used to further localise clone T20782 to YAC clone yUP5C8,the same YAC designated to contain the rsw1 locus in the same chromosomeinterval (FIG. 4).

Furthermore, amplification of the YAC clone yUP5C8 using primers derivedfrom T20782 produces a 500 bp fragment containing two putative exonsidentical to part of the T20782 nucleotide sequence, in addition to twointron sequences.

The cDNA T20782 was considered as a candidate gene involved in cellulosebiosynthesis.

EXAMPLE 4 Nucleotide Sequence Analysis of the cDNA Clone T20782

The nucleotide sequence of the cDNA clone T20782 is presented in SEQ IDNO:1. The nucleotide sequence was obtained using a Dye Terminator CycleSequencing kit (Perkin Elmer cat. #401384) as recommended by themanufacturer. Four template clones were used for nucleotide sequencingto generate the sequence listed. The first template was the cDNA cloneT20782. This template was sequenced using the following sequencingprimers:

-   -   a) 5′-CAATGCATTCATAGCTCCAGCCT-3′ (SEQ ID NO:17)    -   b) 5′-AAAAGGCTGGAGCTATGAATGCAT-3′ (SEQ ID NO:18)    -   c) 5′-TCACCGACAGATTCATCATACCCG-3′ (SEQ ID NO:19)    -   d) 5′-GACATGGAATCACCTTAACTGCC-3′ (SEQ ID NO:20)    -   e) 5′-CCATTCAGTCTTGTCTTCGTAACC-3′ (SEQ ID NO:21)    -   f) 5′-GGTTACGAAGACAAGACTGAAATGG-3′ (SEQ ID NO:22)    -   g) 5′-GAACCTCATAGGCATTGTGGGCTGG-3′ (SEQ ID NO:23)    -   h) 5′-GCAGGCTCTATATGGGTATGATCC-3′ (SEQ ID NO:24)    -   i) Standard M13 forward sequencing primer.    -   j) Standard T7 sequencing primer.

The second template clone (T20782 SphI deletion clone) was constructedby creating a DNA deletion within the T20782 clone. The T20782 clone wasdigested with the restriction enzyme SphI, the enzyme was heat-killed,the DNA ligated and electroporated into NM522 E. coli host cells. TheT20782 SphI deletion clone was then sequenced using a standard M13forward sequencing primer. Two other deletion clones were made for DNAsequencing in a similar fashion but the restriction enzymes EcoRI andSmal were used. The T20782 EcoRI deletion clone and the T20782 Smaldeletion clone were sequenced using a standard T7 sequencing primer. TheDNA sequence shown in SEQ ID NO:1 is for one DNA strand only howeverthose skilled in the art will be able to generate the nucleotidesequence of the complementary strand from the data provided.

The amino acid sequence encoded by clone T20782 was derived and is setforth in SEQ ID NO:2.

The T20782 clone encodes all but the first Aspartate (D) residue of theD, D, D, QXXRW (SEQ ID NO:37) signature conserved in the generalarchitecture of β-glycosyl transferases. In particular, T20782 encodes 5amino acid residues of the D, D, D, QXXRW signature, between amino acidpositions 109 and 370 of SEQ ID NO:2. The conserved Aspartate,Aspartate, Glutamine, Arginine and Tryptophan amino acid residues areshown below, in bold type, with the local amino acid residues alsoindicated:

-   -   1. Amino acid residues 105 to 113 of SEQ ID NO:2:        -   LLNVDCDHY;    -   2. Amino acid residues 324 to 332 of SEQ ID NO:2:        -   SVTEDILTG; and    -   3. Amino acid residues 362 to 374 of SEQ ID NO:2:        -   DRLNQVLRWALGS.

It must be noted that these invariable amino acids merely indicate thatthe T20782 derived amino acid sequence belongs to a very broad group ofglycosyl transferases. Some of these enzymes such as cellulose synthase,chitin synthase, alginate synthase and hyaluronic acid synthase producefunctionally very different compounds.

The presence of the conserved amino acid residues merely indicates thatthe T20782 clone may encode a β-glycosyl transferase protein such as thecellulose gene product, cellulose synthase. The fact that the clonelocalises in the vicinity of a gene involved in cellulose biosynthesisis the key feature which now focus interest on the T20782 clone as acandidate for the RSW1 (cellulose synthase) gene.

The T20782 potentially codes for a cellulose synthase.

EXAMPLE 5 Nucleotide Sequence Analysis of the Genomic Clone 23H12

Clone 23H12 contains approximately 21 kb of Arabidopsis thaliana genomicDNA in the region between the left border and right border T-DNAsequences, and localises to the RSW1 candidate YAC yUP5C8. Clone 23H12was isolated by hybridisation using EST20782 insert DNA, from a genomicDNA library made for plant transformation. Cosmid 12C4 was also shown tohybridize to the cDNA clone T20782, however this cosmid appears tocomprise a partial genomic sequence corresponding to the related Ath-AcDNA sequence set forth in SEQ ID NO:7, for which the correspondingamino acid sequence is set forth in SEQ ID NO:8.

A restriction enzyme map of clone 23H12 is presented in FIG. 5.

Nucleotide sequence of 8411bp of genomic DNA in the binary cosmid clone23H12 was obtained (SEQ ID NO:3) by primer walking along the 23H12template, using a Dye Terminator Cycle Sequencing kit (Perkin Elmer cat.#401384) as recommended by the manufacturer. The following primers atleast, were used for DNA sequencing of the 23H12 clone DNA:

a) cs1-R 5′-CAATGCATTCATAGCTCCAGCCT-3′ (SEQ ID NO:17) b) cs1-F5′-AAAAGGCTGGAGCTATGAATGCAT-3′ (SEQ ID NO:18) c) up5′-AGAACAGCAGATACACGGA-3′ (SEQ ID NO:25) d) ve76-R25′-ATCCGTGTATCTGCTGTTCTTACC-3′ (SEQ ID NO:26) e) est1-R5′-AATGCTCTTGTTGCCAAAGCAC-3′ (SEQ ID NO:27) f) sve76-F5′-ATTGTCCAGCCTTCTTCAGG-3′ (SEQ ID NO:28) g) ve76-R5′-CTGAAGAAGGCTGGACAATGC-3′ (SEQ ID NO:29) h) B12-R15′-AGGTAAGCATAGCTGAACCATC-3′ (SEQ ID NO:30) i) B12-R25′-AGTAGATTGCAGATGGTTTTCTAC-3′ (SEQ ID NO:31) j) B12-R35′-TTCAATGGGTCCACTGTACTAAC-3′ (SEQ ID NO:32) k) B12-R45′-ATTCAGATGCACCATTGTC-3′ (SEQ ID NO:33)

The structure of the RSW1 gene contained in cosmid clone 23H12 is alsopresented in FIG. 5. As shown therein, coding sequences in 23H12, fromthe last 12 bp of exon 7 to the end of exon 14, correspond to the fullT20782 cDNA sequence (i.e. SEQ ID NO:1). The nucleotide sequences of theRSW1 gene comprising exons 1 to 8 were amplified from A.thalianaColumbia double-stranded cDNA, using amplification primers upstream ofthe RSW1 start site and a primer internal to the EST clone T20782.

The exons in the RSW1 gene range from 81 bp to 585 bp in length and all5′ and 3′ intron/exon splice junctions conform to the conserved intronrule.

The RSW1 transcript comprises a 5′-untranslated sequence of at least 70bp in length, a 3243 bp coding region and a 360 bp 3′-untranslatedregion. Northern hybridization analyses indicate that the RSW1transcript in wild-type A. thaliana roots, leaves and inflorescences isapproximately 4.0 kb in length, and that a similar transcript sizeoccurs in mutant tissue (data not shown).

The derived amino acid sequence of the RSW1 polypeptide encoded by thecosmid clone 23H12 (i.e. the polypeptide set forth in SEQ ID NO:6) is1081 amino acids in length and contains the entire D, D, D, QXXRW (SEQID NO:37) signature characteristic of β-glycosyl transferase proteins,between amino acid position 395 and amino acid position 822. Theconserved Aspartate, Glutamine, Arginine and Tryptophan residues areshown below, in bold type, with the local amino acid residues alsoindicated:

-   -   1. amino acid residues 391 to 399 of SEQ ID NO:6:        -   YVSDDGSAM    -   2. Amino acid residues 557 to 565 of SEQ ID NO:6:        -   LLNVDCDHY;    -   3. Amino acid residues 776 to 784 of SEQ ID NO:6:        -   SVTEDILTG; and    -   4. Amino acid residues 814 to 826 of SEQ ID NO:6:        -   DRLNQVLRWALGS.

The second and third conserved Aspartate residues listed supra, and thefourth conserved amino acid sequence motif listed supra (i.e. QVLRW) arealso present in the cDNA clone T20782 (see Example 4 above).

The 23H12 clone potentially encodes a cellulose synthase.

EXAMPLE 6 Complementation of the rsw1 Mutation

The complementation of the cellulose mutant plant rsw1 is the key testto demonstrate the function of the clone 23H12 gene product.Complementation of the rsw1 phenotype was demonstrated by transformingthe binary cosmid clone 23H12, or a derivative clone thereof encoding afunctional gene product, into the Arabidopsis thaliana cellulose mutantrsw1. Two DNA constructs (23H12 and pRSW1) were used to complement thersw1 mutant plant line.

1. Construct 23H12

Clone 23H12 is described in Example 5 and FIG. 5.

2. Construct pRSW1

The 23H12 construct has an insert of about 21 kb in length. Todemonstrate that any complementation of the phenotype of the rsw1mutation is the result of expression of the gene which corresponds toSEQ ID NO:3, a genetic construct, designated as pRSW1, comprising theputative RSW1 gene with most of the surrounding DNA deleted, wasproduced. A restriction enzyme (RE) map of the RSW1 gene insert in pRSW1is provided in FIG. 5.

To produce pRSW1, the RSW1 gene was subcloned from cosmid 23H12 andcloned into the binary plasmid pBIN19. Briefly, Escherichia coli cellscontaining cosmid 23H12 were grown in LB medium supplemented withtetracyclin (3.5 mg/L). Plasmid DNA was prepared by alkaline lysis anddigested sequentially with restriction enzymes PvuII and SalI. Twoco-migrating fragments of 9 kb and 10 kb, respectively, were isolated asa single fraction from a 0.8% (w/v) agarose gel. The RSW1 gene wascontained on the 10 kb PvuII/SalI fragment. The 9 kb fragment appearedto be a PvuII cleavage product not comprising the RSW1 gene. Therestriction fragments were ligated into pBIN19 digested with SmaI andSalI. An aliquot of the ligation mix was introduced by electroporationinto E. coli strain XLB 1. Colonies resistant to kanamycin (50 mg/L)were selected and subsequently characterised by restriction enzymeanalysis to identify those clones which contained only the 10 kbPvuII/SalI fragment comprising the RSW1 gene, in pBIN19.

3. Transfer of the 23H12 and pRSW1 Constructs to Agrobacteriumtumefaciens

Cosmid 23H12 was transferred to Agrobacterium by triparental mating,essentially as described by Ditta et al. (1980). Three bacterial strainsas follows were mixed on solid LB medium without antibiotics: Strain 1was an E. coli helper strain containing the mobilising plasmid pRK2013,grown to stationary phase; Strain 2 was E coli containing cosmid 23H 12,grown to stationary phase; and Strain 3 was an exponential-phase cultureof A. tumefaciens strain AGL1 (Lazo et al., 1991). The mixture wasallowed to grow over night at 28° C., before an aliquot was streaked outon solid LB medium containing antibiotics (ampicillin 50 mg/L,rifampicin 50 mg/L, tetracyclin 3.5 mg/L) to select for transformed A.tumefaciens AGL1. Resistant colonies appeared after 2–3 days at 28° C.and were streaked out once again on selective medium for furtherpurification. Selected colonies were then subcultured in liquid LBmedium supplemented with rifampicin (50 mg/L) and tetracyclin (3.5 mg/L)and stored at −80° C.

Plasmid pRSW1 (initially designated as p2029) was introduced into A.tumefaciens strain AGL1 by electroporation.

4. Transformation of rsw1 Plants

The rsw1 plant line was transformed with constructs 23H12 and pRSW1using vacuum infiltration essentially as described by Bechtold et al.(1993).

5. Analysis of Radial Swelling in Transformants

Complementation of the radial swelling (rsw) phenotype, which ischaracteristic of the rsw1 mutant plant, was assayed by germinatingtransformed (i.e. T1 seed) rsw1 seeds obtained as described supra onHoaglands plates containing 50 μg/ml kanamycin. Plates containing thetransformed seeds were incubated at 21° C. for 10–12 days.Kanamycin-resistant seedlings were transferred to fresh Hoaglands platescontaining 50 μg/ml kanamycin and incubated at 31° C. for 2 days.Following this incubation, the root tip was examined for a radialswelling phenotype. Under these conditions, the roots of wild-typeplants do not show any radial swelling phenotype however, the roots ofrsw1 plants show clear radial swelling at the root tip and also have ashort root compared to the wild-type plants. As a consequence,determination of the radial swelling phenotype of the transformed plantswas indicative of successful complementation of the rsw1 phenotype.

The kanamycin-resistant seedlings were maintained by further growth ofseedlings at 21° C., following the high temperature incubation. Onceplants had recovered, the seedlings were transferred to soil and grownin cabinets at 21° C. (16 hr light/8 hr dark cycle). T2 seed was thenharvested from mature individual plants.

Using the 23H12 construct for rsw1 transformation, a total of 262kanamycin-resistant seedlings were obtained. All of these transformantswere tested for complementation of the root radial swelling phenotype. Atotal of 230 seedlings showed a wild type root phenotype, while only 32seedlings showed the radial swelling root phenotype characteristic ofrsw1 plants. By way of example, FIG. 6 shows the phenotypes oftransformed seedlings compared to untransformed wild-type and rsw1seedlings, following incubation at 31° C. As shown in FIG. 6, there isclear complementation of the radial swelling phenotype in thetransformed seedlings, with normal root length being exhibited by thetransformed seedlings at 31° C.

Using the pRSW1 construct for transformation, a total of 140kanarnycin-resistant seedlings were obtained. All of the 11 seedlingstested for complementation of the root radial swelling phenotype showeda wild type root phenotype and none of the seedlings showed any signs ofradial swelling in the roots.

6. General Morphological Analysis of the Complemented rsw1 Mutant Line

Further characterisation of the complemented rsw1 plants has shown thatother morphological characteristics of rsw1 have also been restored inthe transgenic lines, for example the bolt (inflorescence) height, andthe ability of the plants to grow wild type cotyledons, leaves,trichomes, siliques and flowers at 31° C. (data not shown).

7. Biochemical Complementation of the rsw1 Mutant Line

T2 seed from transformations using cosmid 23H12 as described supra oralternatively, using the binary plasmid pBin19 which lacks any RSW1 genesequences, was sown on Hoagland's solid media containing kanamycin (50μg/ml), incubated for 2 days at 21° C. and then transferred to 31° C.for 5 days. Wild-type A. thaliana Columbia plants were grown undersimilar conditions but without kanamycin in the growth medium. Kanamycinresistant T2 seedlings which have at least one copy of the 23H12 cosmidsequence, and wild-type seedlings, were collected and frozen forcellulose analysis.

Cellulose levels were determined as acetic-nitric acid insolublematerial (Updegraph, 1969) for 10 lines of kanamycin-resistant T2 plantstransformed with the 23H12 cosmid sequence, and compared to thecellulose levels in rsw1 mutant plants, wild-type A. thaliana Columbiaplants and A. thaliana Columbia plants transformed with the binaryplasmid pBin19. The results are provided in Table 7.

As shown in Table 7, the cellulose levels have been significantlyelevated in the complemented rsw1 (T2) plants, compared to the celluloselevels measured in the rsw1 mutant parent plant. In fact, celluloselevels in the 23H 12-transformed plants, expressed relative to the freshweight of plant material or on a per seedling basis, are notsignificantly different from the cellulose levels of either wild-typeArabidopsis thaliana Columbia plants or A. thaliana Columbia transformedwith the binary plasmid pBin19. These data indicate that the 23H12cosmid is able to fully complement the cellulose-deficient phenotype ofthe rsw1 mutant.

Homozygous T3 lines are generated to confirm the data presented in Table7.

Furthermore, data presented in Table 7 indicate that there is nodifference in the rate of growth of the T2 transformed rsw1 plants andwild-type plants at 31° C., because the fresh weight of such plants doesnot differ significantly. In contrast, the fresh weight of mutant rsw1seedlings grown under identical conditions is only approximately 55% ofthe level observed in T2 lines transformed with 23H12 (range about 30%to about 80%). These data support the conclusion that cellulose levelshave been manipulated in the complemented rsw1 (T2) plants.

Furthermore, the rate of cellulose synthesis in 23H12-transformed plantsand wild-type plants at 31° C., as measured by ¹⁴C incorporation is alsodetermined.

Furthermore, the β-1,4-glucan levels and starch levels in the 23H12transformant lines are shown to be similar to the β-1,4-glucan andstarch levels in wild-type plants.

TABLE 7 CELLULOSE LEVELS IN rsw1 PLANTS TRANSFORMED WITH COSMID CLONE23H12 SEEDLING CELLULOSE CELLULOSE SAMPLE SIZE FRESH WEIGHT (mgcellulose/ (mg cellulose/ PLANT LINE (No. of plants) (mg) 100 mg tissue)seedling) 1.2 (rsw1 + 23H12) 126 2.51 1.23 0.031 1.4 (rsw1 + 23H12) 1322.25 2.50 0.056 2.1 (rsw1 + 23H12) 126 3.23 1.29 0.042 3.1 (rsw1 +23H12) 127 3.75 1.23 0.046 3.10 128 3.52 1.69 0.060 (rsw1 + 23H12) 4.4(rsw1 + 23H12) 110 5.14 1.31 0.067 4.5 (rsw1 + 23H12) 125 3.18 1.260.040 5.3 (rsw1 + 23H12) 124 2.77 1.17 0.032 9.2 (rsw1 + 23H12) 125 2.261.41 0.032 10.8 126 2.4 1.20 0.029 (rsw1 + 23H12) Columbia/pBin19 1062.64 1.34 0.035 Columbia 178 2.73 1.18 0.032 rsw1 mutant 179 1.77 0.840.015

EXAMPLE 7 Determination of the Full-Length Nucleotide Sequence Encodingthe Wild-Type RSW1 Polypeptide

Arabidopsis thaliana double-stranded cDNA and cDNA libraries wereprepared using the CAPFINDER cDNA kit (Clontech). RNA was isolated fromwild-type Columbia grown in sterile conditions for 21 days.

Approximately 100,000 cDNA clones in an unamplified cDNA library werescreened under standard hybridization conditions at 65° C., using aprobe comprising ³²P-labeled DNA amplified from double stranded cDNA. Toprepare the hybridization probe, the following amplification primerswere used:

-   -   1. 2280-F:5′GAATCGGCTACGAATTTCCCA 3′ (see SEQ ID NO:3)    -   2. 2370-F:5′TTGGTTGCTGGATCCTACCGG 3′ (see SEQ ID NO:3)    -   3. csp1-R:5′GGT TCT AAA TCT TCT TCC GTC 3′ (see SEQ ID NO:1)        wherein the primer combinations were either 2280-F/csp1-R or        2370-F/csp1-R. The primer 2280-F corresponds to nucleotide        positions 2226 to 2246 in SEQ ID NO:3, upstream of the        translation start site. The primer 2370-F corresponds to        nucleotide positions 2314 to 2334 in SEQ ID NO:3, encoding amino        acids 7 through 13 of the RSW1 polypeptide. The primer csp1-R        comprises nucleotide sequences complementary to nucleotides 588        to 608 of the T20782 clone (SEQ ID NO:1) corresponding to        nucleotides 6120 to 6140 of SEQ ID NO:3. The hybridization        probes produced are approximately 1858 nucleotides in length        (2280-F/csp1-R primer combination) or 1946 nucleotides in length        (2370-F/csp1-R primer combination).

Five hybridizing bacteriophage clones were identified, which wereplaque-purified to homogeneity during two successive rounds ofscreening. Plasmids were rescued from the positively-hybridizingbacteriophage clones, using the Stratagene excision protocol for theZapExpress™ vector according to the manufacturer's instructions. Colonyhybridizations confirmed the identity of the clones.

Isolated cDNA clones were sequenced by primer walking similar to themethod described in Examples 4 and 5 supra.

A full-length wild-type RSW1 nucleotide sequence was compiled from thenucleotide sequences of two cDNA clones. First, the 3′-end of the cDNA,encoding amino acids 453–1081 of RSW1, corresponded to the nucleotidesequence of the EST clone T20782 (SEQ ID NO:1). The remaining cDNAsequence, encoding amino acids 1–654 of RSW1, was generated byamplification of the 5′-end from cDNA, using primer 2280-F, whichcomprises nucleotide sequences approximately 50–70 bp upstream of theRSW1 translation start site in cosmid 23H12, and primer csp1-R, whichcomprises nucleotide sequences complementary to nucleotides 588 to 608of the T20782 clone (SEQ ID NO:1).

Several amplified clones are sequenced to show that no nucleotide errorswere introduced by the amplification process. The 5′ and 3′ nucleotidesequences are spliced together to produce the complete RSW1 open readingframe and 3′-untranslated region provided in SEQ ID NO:5.

Those skilled in the art will be aware that the 5′-end and 3′-end of thetwo incomplete cDNAs are spliced together to obtain a full-length cDNAclone, the nucleotide sequence of which is set forth in SEQ ID NO:5.

Of the remaining cDNA clones, no isolated cDNA clone comprised anucleotide sequence which precisely matched the nucleotide sequence ofthe RSW1 gene present in cosmid 23H12. However, several clonescontaining closely-related sequences were obtained, as summarised inTable 8. The nucleotide sequences of the Ath-A and Ath-B cDNAs areprovided herein as SEQ ID Nos:7 and 9, respectively.

TABLE 8 CHARACTERISATION OF A. thaliana cDNA CLONES CLONE NAMEDESCRIPTION LENGTH SEQ ID NO: RSW1/1A chimeric clone partial notprovided RSW1A chimeric clone partial not provided Ath-A 12C4 cDNAfull-length SEQ ID NO:7 Ath-B new sequence full-length SEQ ID NO:9 RSW4Aidentical to full-length not provided Ath-B

The derived amino acid sequences encoded by the cDNAs listed in Table 8,is provided in FIGS. 8 and 9 and SEQ ID Nos:8 and 10 herein.

FIG. 10 a schematic representation of the important features of the RSW1polypeptide which are conserved within A. thaliana and between A.thaliana and other plant species. In addition to the species indicatedin FIG. 10, the present inventors have also identified maize, wheat, andbarley and Brassica spp. cellulose biosynthetic genes by homologysearch. Accordingly, the present invention extends to cellulose genesand cellulose biosynthetic polypeptides as hereinbefore defined, derivedfrom any plant species, including A. thaliana, cotton, rice, wheat,barley, maize, Eucalyptus spp., Brassica spp. Pinus spp., Populus spp.,Picea spp., hemp, jute and flax, amongst others.

EXAMPLE 8 Isolation of Full-Length Nucleotide Sequence Encoding theMutant RSW1 Polypeptide

Arabidopsis thaliana double-stranded cDNA and cDNA libraries wereprepared using the CAPFINDER cDNA kit (Clontech). RNA was isolated fromArabidopsis thaliana Columbia rsw1 mutant plants grown in sterileconditions for 21 days.

The full-length rsw1 mutant nucleotide sequence was generated bysequencing two amplified DNA fragments spanning the rsw1 mutant gene.The 5′-end sequence of the cDNA (comprising the 5′-untranslated regionand exons 1–11) was amplified using the primer combination 2280-F/csp1-R(Example 7). The 3′-end sequence was amplified using the primers EST1-Fand cs3-R set forth below:

-   -   1. Primer EST1-F: 5′AATGCTTCTTGTTGCCAAAGCA 3′ (see SEQ ID NO:5)    -   2. Primer cs3-R: 5′GACATGGAATCACCTTAACTGCC 3′ (see SEQ ID NO:5)        wherein primer EST1-F corresponds to nucleotide positions        1399–1420 of SEQ ID NO:5 (within exon 8) and primer cs3-R is        complementary to nucleotides 3335–3359 of SEQ ID NO:5 (within        the 3′-untranslated region of the wild-type transcript).

The full-length sequence of the mutant rsw1 transcript is set forthherein as SEQ ID NO:11.

Whilst not being bound by any theory or mode of action, a singlenucleotide substitution in the rsw1 mutant nucleotide sequence(nucleotide position 1716 in SEQ ID NO:11), relative to the wild-typeRSW1 nucleotide sequence (nucleotide position 1646 in SEQ ID NO:5),resulting in Ala549 being substituted with Val549 in the mutantpolypeptide, may contribute to the altered activity of the RSW1polypeptide at non-permissive temperatures such as 31° C. Additionalamino acid substitutions are also contemplated by the present invention,to alter the activity of the RSW1 polypeptide, or to make thepolypeptide temperature-sensitive.

EXAMPLE 9 Antisense Inhibition of Cellulose Production in TransgenicPlants

1. Construction of an Antisense RSW1 Binary Vector

One example of transgenic plants in which cellulose production isinhibited is provided by the expression of an antisense geneticconstruct therein. Antisense technology is used to target expression ofa cellulose gene(s) to reduce the amount of cellulose produced bytransgenic plants.

By way of exemplification, an antisense plant transformation constructhas been engineered to contain the T20782 cDNA insert (or a partthereof) in the antisense orientation and in operable connection withthe CaMV 35S promoter present in the binary plasmid pRD410 (Datla et al.1992). More particularly, the T20782 cDNA clone, which comprises the3′-end of the wild-type RSW1 gene, was digested with XbaI and KpnI andcloned into the kanamycin-resistant derivative of pGEM3zf(−), designatedas plasmid, pJKKMf(−). The RSW1 sequence was sub-cloned, in theantisense orientation, into the binary vector pRD410 as a XbaI/SacIfragment, thereby replacing the β-glucuronidase (GUS or uidA) gene. Thisallows the RSW1 sequence to be transcribed in the antisense orientationunder the control of the CaMV 35S promoter.

The antisense RSW1 binary plasmid vector was transferred toAgrobacterium tumefaciens strain AGL1, by triparental mating andselection on rifampicin and kanamycin, as described by Lazo et al.(1991). The presence of the RSW1 insert in transformed A. tumefacienscells was confirmed by Southern hybridization analysis (Southern, 1975).The construct was shown to be free of deletion or rearrangements priorto transformation of plant tissues, by back-transformation intoEscherichia coli strain JM 101 and restriction digestion analysis.

2. Transformation of Arabidopsis thaliana

Eight pots, each containing approximately 16 A. thaliana ecotypeColumbia plants, were grown under standard conditions. Plant tissue wastransformed with the antisense RSW1 binary plasmid by vacuuminfiltration as described by Bechtold et al. (1993). Infiltration mediacontained 2.5% (w/v) sucrose and plants were infiltrated for 2 min untila vacuum of approximately 400 mm Hg was obtained. The vacuum connectionwas shut off and plants allowed to sit under vacuum for 5 min.

Approximately 34,000 T1 seed was screened on MS plates containing 50μg/ml kanamycin, to select for plants containing the antisense RSW1construct. Of the T1 seed sown, 135 kanamycin-resistant seedlings wereidentified, of which 91 were transferred into soil and grown at 21° C.under a long-day photoperiod (16 hr light; 8 hr dark).

Of the 91 transgenic lines, 19 lines were chosen for further analysiswhich had anther filaments in each flower which were too short todeposit pollen upon the stigma and, as a consequence, requiredhand-pollination to obtain T2 seed therefrom.

T2 seed from 14 of these 19 lines was plated out onto vertical Hoaglandsplates containing kanamycin to determine segregation ratios. Betweenfive and ten seed were plated per transgenic line. Control seeds,including A. thaliana Columbia containing the binary vector pBIN19(Bevan, 1984) and segregating 3:1 for kanamycin resistance, and the rsw1mutant transformed with the NPTII gene, also segregating 3:1 forkanamycin resistance, were grown under the same conditions.Kanamycin-resistant plants were transferred to soil and grown at 21° C.under long days, until flowering. Untransformed Arabidopsis thalianaColumbia plants were also grown under similar conditions, in the absenceof kanamycin.

3. Morphology of Antisense-RSW1 Plants

A comparison of the morphology of antisense RSW1 plants grown at 21° C.,to mutant rsw1 plants grown at the non-permissive temperature (i.e. 31°C.) has identified a number of common phenotypes. For example, theantisense plants exhibit reduced fertility, inflorescence shortening andhave short anthers, compared to wild-type plants, when grown at 21° C.These phenotypes are also observed in mutant rsw1 plants grown at 31° C.These results suggest that the antisense construct in the transgenicplants may be targeting the expression of the wild-type RSW1 gene at 21°C.

FIG. 7 shows the reduced inflorescence (bolt) height in antisense35S-RSW1 plants compared to wild-type A. thaliana Columbia plants grownunder identical conditions.

4. Cell Wall Carbohydrate Analysis of Antisense Plants.

T3 plants which are homozygous for the 35S-RSW1 antisense construct aregenerated and the content of cellulose therein is determined asdescribed in Example 1. Plants expressing the antisense construct areshown to have significantly less cellulose in their cell walls, comparedto wild-type plants. Additionally, the levels of non-crystallineβ-1,4-glucan and starch are elevated in the cells of antisense plants,compared to otherwise isogenic plant lines which have not beentransformed with the antisense genetic construct.

5. Antisense 35S-RSW1 mRNA Expression Levels in Transgenic Plants

Total RNA was extracted from 0.2 g of leaf tissue derived from 33kanamycin-resistant T1 plants containing the antisense 35S-RSW1 geneticconstruct, essentially according to Longemann et al. (1986). Total RNA(25 μg) was separated on a 2.2M formaldehyde/agarose gel, blotted ontonylon filters and hybridized to a riboprobe comprising the sense strandsequence of the cDNA clone T20782. To produce the riboprobe, T7 RNApolymerase was used to transcribe sense RNA from a linearised plasmidtemplate containing T20782, in the presence of [α-³²P]UTP.Hybridizations and subsequent washes were performed as described byDolferus et al. (1994). Hybridized membranes were exposed to Phosphorscreens (Molecular Dynamics, USA).

The levels of expression of the RSW1 antisense transcript weredetermined and compared to the level of fertility observed for the plantlines. As shown in Table 9, the level of antisense gene expression iscorrelated with the reduced fertility phenotype of the antisense plants.In 13 lines, a very high or high level of expression of the 35S-RSW1antisense gene was observed and, in 11 of these lines fertility wasreduced. Only lines 2W and 3E which expressed high to very high levelsof antisense mRNA, appeared to be fully fertile. In 12 lines whichexpressed medium levels of antisense mRNA, approximately one-half werefertile and one-half appeared to exhibit reduced fertility. In contrast,in 8 plant lines in which only a low or very low level of expression ofthe antisense 35S-RSW1 genetic construct was observed, a wild-type (i.e.fertile) phenotype was observed for all but one transgenic line, line2R.

Data presented in Table 9 and FIG. 7 indicate that the phenotype of thecellulose-deficient mutant rsw1 may be reproduced by expressingantisense RSW1 genetic constructs in transgenic plants.

To confirm reduced cellulose synthesis and/or deposition in transgenicplants expressing the antisense RSW1 gene, the level of cellulose ismeasured by the ¹⁴C incorporation assay or as acetic/nitric acidinsoluble material as described in Example 1 and compared to celluloseproduction in otherwise isogenic wild-type plants. Cellulose productionin the transgenic plants is shown to be significantly reduced comparedto wild-type plants. The severity of phenotype of the transgenic plantsthus produced varies considerably, depending to some extent upon thelevel of inhibition of cellulose biosynthesis.

TABLE 9 LEVELS OF ANTISENSE GENE EXPRESSION AND FERTILITY IN T1 LINES OFANTISENSE 35S-RSW1 PLANTS T1 ANTISENSE PLANT 35S-RSW1 LINE EXPRESSIONFERTILITY B very high sterile* 2B very high sterile* 3E very highfertile 2E high sterile* 2K high sterile* 2M high sterile* 2O highsterile* 2P high sterile* 2W high fertile 2Z high sterile* 3G highsterile* 3Q high sterile* 7Q high sterile* 7N medium sterile* 7G mediumfertile 1C medium sterile* 2X medium sterile* 2H medium fertile C mediumsterile* F medium sterile* 2Q medium fertile 3P medium sterile* 3Tmedium fertile 5D medium sterile* 6A medium fertile 8E low fertile 2Rlow sterile* 7A low fertile 7S low fertile 7O low fertile 7R low fertile1B very low fertile 2U very low fertile *sterile phenotype notindicative of complete sterility, but that hand pollination at least, isrequired to obtain seed from such plants.

EXAMPLE 10 RSW1 Related Sequences in Rice Plants

To identify RSW1 related nucleotide sequences in rice, a geneticsequence database was searched for nucleotide sequences which wereclosely-related to one or more of the Arabidopsis thaliana RSW1nucleotide sequences described in the preceding Examples. Rice EST S0542(MAFF DNA bank, Japan) was identified, for which only a partialnucleotide sequences was available. Additionally, before the instantinvention, there was no probable function attached to the rice EST S0542sequence.

The present inventors have obtained the complete nucleotide sequence ofclone S0542 and derived the amino acid sequence encoded therefor. TheS0542 cDNA is only 1741 bp in length and appears to be a partial cDNAclone because, although it comprises 100 bp of 5′-untranslated sequenceand contains the ATG start codon, it is truncated at 3′-end and, as aconsequence encodes only the first 547 amino acid residues of the riceRSW1 or RSW1-like polypeptide. Based upon the length of thecorresponding Arabidopsis thaliana RSW1 polypeptide (1081 amino acids),the rice RSW1 sequence set forth in SEQ ID NO:14 appears to containapproximately one-half of the complete amino acid sequence.

The N-terminal half of the rice RSW1 amino acid sequence isapproximately 70% identical to the Arabidopsis thaliana RSW1 polypeptideset forth in SEQ ID NO:6, with higher homology (approximately 90%)occurring between amino acid residues 271–547 of the rice sequence.These data strongly suggest that S0542 is the rice homologue of the A.thaliana RSW1 gene. Alignments of rice, A. thaliana and cotton RSW1amino acid sequences are presented in FIGS. 9 and 10.

To isolate full-length cDNA clones and genomic clone equivalents ofS0542 (this study and MAFF DNA bank, Japan) or D48636 (Pear et al.,1996), cDNA and genomic clone libraries are produced using rice mRNA andgenomic DNA respectively, and screened by hybridisation using the S0542or D48636 cDNAs as a probe, essentially as described herein.Positive-hybridising plaques are identified and plaque-purified, duringfurther rounds of screening by hybridisation, to single plaques.

The rice clones are sequenced as described in the preceding Examples todetermine the complete nucleotide sequences of the rice RSW1 genes andderived amino acid sequences therefor. Those skilled in the art will beaware that such gene sequences are useful for the production oftransgenic plants, in particular transgenic cereal plants having alteredcellulose content and/or quality, using standard techniques. The presentinvention extends to all such genetic sequences and applicationstherefor.

EXAMPLE 11 RSW1 Related Sequences in Cotton Plants

A ³²P-labeled RSW1 PCR fragment was used to screen approximately 200,000cDNA clones in a cotton fibre cDNA library. The RSW1 PCR probe wasinitially amplified from Arabidopsis thaliana wild type cDNA using theprimers 2280-F and csp1-R described in the preceding Examples, and thenre-amplified using the primer combination 2370-F/csp1-R, also describedin the preceding Examples.

Hybridisations were carried out under low stringency conditions at 55°C.

Six putative positive-hybridising plaques were identified in the firstscreening round. Using two further rounds of screening by hybridisation,four of these plaques were purified to single plaques. Three plaqueshybridise very strongly to the RSW1 probe while the fourth plaquehybridises less intensely.

We conclude that the positive-hybridising plaques which have beenpurified are strong candidates for comprising cotton RSW1 gene sequencesor RSW1-like gene sequences. Furthermore, the cotton cDNAs may encodethe catalytic subunit of cellulose synthase, because the subunit proteinarchitecture of cellulose synthase appears to be highly conserved amongplants as highlighted in the preceding Example.

Furthermore, a Southern blot of cotton genomic DNA digested with BglIIwas hybridised with the 5′ end of the RSW1 cDNA, under low stringencyhybridisation conditions at 55° C. Results are presented in FIG. 11.These data demonstrate that RSW1-related sequences exist in the cottongenome.

The cotton cDNA clones described herein are sequenced as described inthe preceding Examples and used to produce transgenic cotton plantshaving altered fibre characteristics. The cDNAs are also used togenetically alter the cellulose content and/or quality of other plants,using standard techniques.

EXAMPLE 12 RSW1 Related Sequences in Eucalyptus SPP.

Putative Eucalyptus spp. cellulose synthase catalytic subunit genefragments were obtained by amplification using PCR. DNA primers weredesigned to conserved amino acid residues found in the Arabidopsisthaliana RSW1 and 12C4 amino acid sequences. Three primers were used forPCR. The primers are listed below:

-   pcsF-I 5′-A A/G A A G A T I G A C/T T A C/T C/T T I A A A/G G A C/T    A A-3′(SEQ ID NO:34)-   pcsR-II 5′-A T I G T I G G I G T I C G/T A/G T T C/T T G A/T/G/C C    T/G A/T/C/G C C-3′ (SEQ ID NO:35)-   pcsF-115′-G C I A T G A A A/G A/C G I G A I T A C/T G A A/G G    A-3′(SEQ ID NO:36)

Using standard PCR conditions (50° C. annealing temperature) andsolutions, the primer sets pcsF-I/pcsR-II and pcsF-II/pcsR-II were usedto amplify genetic sequences from pooled Eucalyptus spp. cDNA. In thefirst reaction primers pcsF-I and pcsR-II were used to generate afragment approximately 700 bp in length. In the second PCR reaction,which used primers pcsF-II and pcsR-II, a fragment estimated to 700 bpwas obtained. The sizes of the PCR fragments are within the size rangeestimated for the corresponding Arabidopsis thaliana sequences.

We conclude that the amplified Eucalyptus spp. PCR fragments are likelyto be related to the Arabidopsis thaliana RSW1 gene and may encode atleast a part of the Eucalyptus spp. cellulose synthase catalyticsubunit.

The Eucalyptus spp. PCR clones described herein are sequenced asdescribed in the preceding Examples and used to isolate thecorresponding full-length Eucalyptus spp. cDNAs and genomic geneequivalents. Those skilled in the art will be aware that such genesequences are useful for the production of transgenic plants, inparticular transgenic Eucalyptus spp. plants having altered cellulosecontent and/or quality, using standard techniques. The present inventionextends to all such genetic sequences and applications therefor.

EXAMPLE 13 Non-Crystalline B-1,4-Glucan as a Modifier of Cell WallProperties

The properties of plant cell walls depend on the carbohydrates, proteinsand other polymers of which they are composed and the complex ways inwhich they interact. Increasing the quantities of non-crystallineβ-1,4-glucan in cell walls affects those wall properties which influencemechanical, nutritional and many other qualities as well as havingsecondary consequences resulting from the diversion of carbon intonon-crystalline glucan at the expense of other uses. To illustrate oneof these effects, we investigated the ability of the non-crystallineglucan to hydrogen bond to other wall components particularly cellulosein the way that has been shown to be important for wall mechanics.

Hemicelluloses such as xyloglucans cross-link cellulose microfibrils byhydrogen bonding to the microfibril surface (Levy et al, 1991). Sincethe β-1,4-glucan backbone of xyloglucan is thought to be responsible forhydrogen bonding (with the xylose, galactose and fucose substitutionslimiting the capacity to form further hydrogen bonds) we can expect thenon-crystalline β-1,4-glucan also to have a capacity to hydrogen bondand cross link cellulose. The effectiveness of strong alkalis inextracting xyloglucans is thought to relate to their disruption of thehydrogen bonds with cellulose (Hayashi and MacLachlan, 1984).

To demonstrate that the non-crystalline β-1,4-glucan forms similarassociations with the cellulose microfibrils, we examined whether the 4M KOH fraction, extracted from shoots of the rsw1 mutant and from wildtype RSW1 plants, contained non-crystalline glucan in addition toxyloglucan. The non-crystalline glucan was separated from xyloglucan inthe 4 M KOH extract by dialysing the neutralised extract againstdistilled water and centrifuging at 14000 g for 1 hour. The pellet wasshown to be a pure β-1,4-glucan by using the methods for monosaccharideanalysis, methylation analysis and enzyme digestion used to characterisethe glucan in the ammonium oxalate fraction (see Example 1).

Table 10 shows the presence of substantial quantities of glucanrecovered in pure form in the pellet from 4 M KOH fractions extractedfrom the overproducing rsw1 mutant of Arabidopsis thaliana. These dataalso demonstrate the presence of smaller quantities of non-crystallineβ-1,4-glucan in the 4 M KOH fraction from wild type plants, compared torsw1, particularly when grown at 31° C.

TABLE 10 Glucose contents* of 4 M KOH fractions from shoots of wild-type and rsw1mutant Arabidopsis thaliana plants wild-type rsw1 mutantGlucose fraction 21° C. 31° C. 21° C. 31° C. xyloglucan andnon-crystall- 36.4 56.9 27.1 93.1 ine glucan in whole extractnon-crystalline glucan in 7.8 20.5 7.6 56.0 pellet *, nmol glucose/mgplant dry weight after TFA hydrolysis

The monosaccharide composition of the supernatant remaining aftercentrifugation was determined after TFA hydrolysis. These data, and datafrom methylation analysis, are consistent with the supernatant being arelatively pure xyloglucan. The supernatant was free of glucan, becauseno glucose could be released by the endocellulase/-glucosidase mixturethat released glucose from β-1,4-glucan.

The presence of both non-crystalline β-1,4-glucan and xyloglucan in the4 M KOH fraction, when taken together with the implications fromstructural predictions (Levy et al. 1991), is consistent with some ofthe non-crystalline β-1,4-glucan in the wall hydrogen bonding tocellulose microfibrils in similar fashion to the β-1,4-glucan backboneof xyloglucan.

The cross linking provided when xyloglucans and other hemicellulosesbind to two or more microfibrils is an important determinant of themechanical properties of cellulosic walls (Hayashi, 1989). The effectsof increasing the amounts of non-crystalline β-1,4-glucan in walls arelikely to be greatest in walls which otherwise possess relatively lowlevels of cross linking as a result of high ratios of cellulose:hemicelluloses. Such conditions are common in secondary walls includingthose of various fibres, and the cellulose:hemicellulose ratio isparticularly high in cotton fibres.

The effects on wall mechanical properties of overproducingnon-crystalline glucan are shown by transforming plants with the mutantallele of rsw1 (SEQ ID NO:11) operably under the control of either theRSW1 promoter derived from SEQ ID NO:3 or SEQ ID NO:4 or alternatively,an appropriate constitutive promoter such as the CaMV 35S promoter.Production of non-crystalline glucan is quantified by fractionating thecell walls using the methods described above to show in particular thatnon-crystalline glucan is recovered in the 4 M KOH fraction. Mechanicalproperties of the cell walls are measured using standard methods forfibre analysis to study parameters such as stress-strain curves, andbreaking strain, amongst other properties.

EXAMPLE 14 Over-Expression of Cellulose Synthase in Transgenic Plants

Three strategies are employed to over-express cellulose synthase inArabidopsis thaliana plants.

In the first strategy, the CaMV 35S promoter sequence is operablyconnected to the full-length cellulose synthase cDNA which is obtainableby primer extension of SEQ ID NO:1. This is achievable by cloning thefull-length cDNA encoding cellulose synthase, in the sense orientation,between the CaMV 35S promoter or other suitable promoter operable inplants and the nopaline synthase terminator sequences of the binaryplasmid pBI121.

In the second strategy, the coding part of the genomic gene is cloned,in the sense orientation, between the CaMV 35S promoter and the nopalinesynthase terminator sequences of the binary plasmid pBI121.

In the third strategy, the 23H12 binary cosmid clone or the derivativepRSW1, containing the cellulose synthase gene sequence operably underthe control of the cellulose synthase gene promoter and terminatorsequences is prepared in a form suitable for transformation of planttissue.

For Agrobacterium-mediated tissue transformation, binary plasmidconstructs discussed supra are transformed into Agrobacteriumtumefaciens strain AGL 1 or other suitable strain. The recombinant DNAconstructs are then introduced into wild type Arabidopsis thalianaplants (Columbia ecotype), as described in the preceding Examples.

Alternatively, plant tissue is directly transformed using the vacuuminfiltration method described by Beshtold et al. (1993).

The transgenic plants thus produced exhibit a range of phenotypes,partly because of position effects and variable levels of expression ofthe cellulose synthase transgene.

Cellulose content in the transgenic plants and isogenic untransformedcontrol plants is determined by the ¹⁴C incorporation assay or asacetic/nitric acid insoluble material as described in Example 1. Ingeneral, the level of cellulose deposition and rates of cellulosebiosynthesis in the transgenic plants are significantly greater than foruntransformed control plants.

Furthermore, in some cases, co-supression leads to mimicry of the rsw1mutant phenotype.

EXAMPLE 15 Site-Directed Mutagenesis of the RSW1 Gene

The nucleotide sequence of the RSW1 gene contained in 23H12 is mutatedusing site-directed mutagenesis, at several positions to alter itscatalytic activity or substrate affinity or glucan properties. In oneexample, the RSW1 gene is mutated to comprise one or more mutationspresent in the mutant rsw1 allele.

The mutated genetic sequences are cloned into binary plasmid describedin the preceding Examples, in place of the wild-type sequences. Planttissue obtained from both wild-type Arabidopsis thaliana (Columbia)plants and A. thaliana rsw1 plants is transformed as described hereinand whole plants are regenerated.

Control transformations are performed using the wild-type cellulosesynthase gene sequence.

EXAMPLE 16 Phenotypes of Plants Expressing Mutated RSW1 Genes

Plants transformed with genetic constructs described in Example 15 (andelsewhere) are categorised initially on the basis of number of transgenecopies, to eliminate variability arising therefrom. Plants expressingsingle copies of different transgenes are analysed further for cell wallcomponents, including cellulose, non-crystalline β-1,4-glucan polymer,starch and carbohydrate content.

1. Cellulose Content

Cellulose content in the transgenic plants is determined by the ¹⁴Cincorporation assay as described in Example 1. Cell walls are prepared,fractionated and the monosaccharide composition of individual fractionsdetermined as in Example 1.

2. Non-Crystalline β-1,4-Glucan Content

Transgenic plants expressing the rsw1 mutant allele exhibit a higherlevel of non-crystalline, and therefore extractable, β-1,4-glucan incell walls compared to plants expressing an additional copy of thewild-type RSW1 allele. Thus, it is possible to change the crystallinityof the p-1,4-glucan chains present in the cell wall by mutation of thewild-type RSW1 allele.

3. Starch Content

Transgenic plants are also analysed to determine the effect ofmutagenesis of the RSW1 gene on the level of starch deposited in theirroots. The quantity of starch present in material prepared from thecrude wall fraction is determined using the anthrone/H₂SO₄ methoddescribed in Example 1. The data show that mutating the RSW1 gene to themutant rsw1 allele increases starch deposition. This demonstrates thatthe gene can be used to alter the partitioning of carbon intocarbohydrates other than cellulose.

4. Cell Wall Composition

The cell wall composition of transgenic plant material is also analysed.Wild type and rsw1 and transgenic seedlings are grown for 2 d at 21° C.and then kept for a further 5 d at either 21° C. or 31° C. With transferto 31° C. when the seed has scarcely germinated, the wall composition atfinal harvest largely reflects the operation of the mutated rsw1 geneproduct at its restrictive temperature. Cell wall fractionation iscarried out in similar fashion to that described for the ¹⁴C-experiment(Example 1) and the monosaccharide composition of each fraction isquantified by GC/MS after hydrolysis with trifluoroacetic acid or, inthe case of crystalline cellulose, H₂SO₄.

In some transgenic plants in which the RSW1 gene is mutated, themonosaccharide composition is comparable to that observed for homozygousrsw1 plants, at least in some cases, confirming that there is a majorreduction in the quantity of crystalline cellulose in the final, acidinsoluble fraction. Thus, mutation of the RSW1 gene can be performed toproduce changes in the composition of plant cell walls.

EXAMPLE 17 Chemical Modification of the RSW1 Gene to ManipulateCellulose Production and Plant Cell Wall Content.

As demonstrated in the preceding Examples, the RSW1 gene is involved incellulose production and the manipulation of cell wall content.

In the present Example, to identify novel phenotypes and gene sequencesimportant for the normal functioning of the cellulose synthase gene, theRSW1 gene is modified in planta, using the chemical mutagen EMS. Themutant plants are identified following germination and the modified RSW1genes are isolated and characterised at the nucleotide sequence level. Asequence comparison between the mutant gene sequences and the wild typesequence reveals nucleotides which encode amino acids important to thenormal catalytic activity of the cellulose synthase enzyme, at least inArabidopsis thaliana plants.

This approach thus generates further gene sequences of utility in themodification of cellulose content and properties in plants.

EXAMPLE 18 Discussion

Five pieces of evidence make a compelling case that the RSW1 geneproduct encodes the catalytic subunit of cellulose synthase:

-   1. The rsw1 mutation selectively inhibits cellulose synthesis and    promotes accumulation of a non-crystalline β-1,4-glucan;-   2. The rsw1 mutation removes cellulose synthase complexes from the    plasma membrane, providing a plausible mechanism for reduced    cellulose accumulation and placing the RSW1 product either in the    complexes or interacting with them;-   3. The D,D,D,QXXRW (SEQ ID NO:37) signature identifies the RSW1 gene    product as a processive glycosyl transferase enzyme (Saxena, 1995);-   4. The wild type allele corrects the temperature sensitive phenotype    of the rsw1 mutant; and-   5. Antisense expression of the RSW1 in transgenic plants grown at    21° C. reproduces some of the phenotype of rsw1 which is observed    following growth at 31° C.

Consistent with the plasma membrane location expected for a catalyticsubunit, the putative 122 kDa RSW1 product contains 8 predictedmembrane-spanning regions. Six of these regions cluster near theC-terminus (FIG. 10), separated from the other two by a domain that isprobably cytoplasmic and has the weak sequence similarities toprokaryotic glycosyl transferases (Wong, 1990; Saxena, 1990; Matthyse,1995; Sofia, 1994; Kutish, 1996).

RSW1 therefore qualifies as a member of the large family of Arabidopsisthaliana genes whose members show weak similarities to bacterialcellulose synthase. RSW1 is the first member of that family to berigorously identified as an authentic cellulose synthase. Among thediverse genes in A. thaliana, at least two genes show very strongsequence similarities to the RSW1 gene and are most likely members of ahighly conserved sub-family involved in cellulose synthesis. The closelyrelated sequences come from cosmid 12C4, a partial genomic clonecross-hybridising with EST T20782 designated Ath-A, and from a fulllength cDNA designated Ath-B.

Ath-A resembles RSW1 (SEQ ID NO:5) at its N-terminus whereas Ath-Bstarts 22 amino acid residues downstream [FIG. 8 and FIGS. 9A–9J].Closely related sequences in other angiosperms are the rice EST S0542[FIGS. 9A–9J], which resembles the polypeptides encoded by RSW1 andAth-A and the cotton celA1 gene (Pear, 1996) at the N-terminus.

The Arabidopsis thaliana, rice and cotton genes have regions of veryhigh sequence similarity interspersed with variable regions (FIGS. 9A–9Jand 10). Most of the highest conservation among those gene productsoccurs in their central cytoplasmic domain where the weak similaritiesto the bacterial cellulose synthase occur. The N-terminal region thatprecedes the first membrane spanning region is probably also cytoplasmicbut shows many amino acid substitutions as well as sequences in RSW1that have no counterpart in some of the other genes as already noted forcelA. An exception to this is a region comprising 7 cysteine residueswith highly conserved spacings (FIG. 10). This is reminiscent of regionssuggested to mediate protein-protein and protein-lipid interactions indiverse proteins including transcriptional regulators and may accountfor the striking sequence similarity between this region of RSW1 and twoputative soybean bZIP transcription factors (Genbank SOYSTF1A and 1B).

In conclusion, the chemical and ultrastructural changes seen in thecellulose-deficient mutant combine with gene cloning and complementationof the mutant to provide strong evidence that the RSW1 locus encodes thecatalytic subunit of cellulose synthase. Accumulation of non-crystallineβ-1,4-glucan in the shoot of the rsw1 mutant suggests that propertiesaffected by the mutation are required for glucan chains to assemble intomicrofibrils. Whilst not being bound by any theory or mode of action, akey property may be the aggregation of catalytic subunits into plasmamembrane rosettes. At the restrictive temperature, mutant synthasecomplexes disassemble to monomers (or smaller oligomers) that areundetectable by freeze etching. At least in the shoot, the monomers seemto remain biosynthetically active but their β-1,4-glucan products failto crystallise into microfibrils probably because the chains are growingfrom dispersed sites. Crystallisation into microfibrils, with all itsconsequences for wall mechanics and morphogenesis, therefore may dependupon catalytic subunits remaining aggregated as plasma membranerosettes.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

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1. An isolated nucleic acid molecule comprising a coding sequence whichencodes a cellulose synthase polypeptide of a plant, or a complementarysequence to said coding sequence, wherein said coding sequence encodes apolypeptide which has at least 90% sequence identity to SEQ ID NO:6. 2.An isolated nucleic acid molecule which encodes a cellulose synthasepolypeptide of a plant, wherein said polypeptide comprises SEQ ID NO:6.3. A genetic construct which comprises the isolated nucleic acidmolecule according to claim 1 operably linked to a promoter sequencethat is operable in a plant.
 4. A genetic construct which comprises theisolated nucleic acid molecule according to claim 2 operably linked to apromoter sequence that is operable in a plant.
 5. The genetic constructaccording to claim 3 or 4, wherein the nucleic acid molecule is operablyconnected to the promoter sequence in the sense orientation such that acellulose synthase polypeptide or RNA encoding said cellulose synthasepolypeptide is produced when said nucleic acid molecule is expressed ina plant cell containing said genetic construct.
 6. The genetic constructaccording to claim 4, wherein the promoter is the CaMV 35S promoter orthe Arabidopsis thaliana RSW1 gene promoter.
 7. A transgenic planttransformed with a genetic construct according to claim
 3. 8. Atransgenic plant transformed with a genetic construct according to claim4.
 9. A transgenic plant transformed with an isolated nucleic acidmolecule according to claim 1 or 2.